The Puzzle of Ageing - Leibniz Institute for Age Research

The Puzzle of Ageing 

Leibniz Institute for Age Research – 

Fritz Lipmann Institute (FLI) 

Jena, Germany

2 Contents 

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4 Preface by Peter Herrlich 

6 Fritz Lipmann – Biochemist, 

Noble Prize Laureate and 

Pioneer of Age Research 

7 Solving the Puzzle of Ageing 

8 Research Concept: Many Roads – 

One Goal 

Selected Topics 

TUMOUR BIOLOGY 

12 How Fatal Growth Stimuli Are Blocked 

GENOME ANALYSIS 

15 Huddling Together for Safety: 

How Cells Cooperate 

BIOPHYSICS 

18 Virus-Induced Cancer: 

The Structural Basis of 

Viral Cancerogenesis 

GENETICS OF AGEING 

21 What the Turquoise Killifish Can Tell Us 

about Ageing 

BIOINFORMATICS 

24 Analysis and Interpretation of 

Complex Data 

Our Laboratories 

CALKHOVEN LAB 

26 Translational Control of Gene Expression 

CELLERINO LAB 

29 Using a Short-Lived Fish to Investigate 

the Biological Mechanisms Controlling 

Lifespan 

DIEKMANN LAB 

31 Early Ageing and Premature Death: 

When Cellular Control Systems Fail 

ENGLERT LAB 

34 From Genes to Organs: 

How Genes Control Development 

FäNDRICH LAB 

37 Structure and Formation of Amyloid Fibrils 

GöRLACH LAB 

40 Biomolecular Matchmaking: 

How Molecules Contact Each Other 

GREULICH LAB 

43 Getting Sorted: 

Functional Molecule Blocks 

GROSSE LAB 

46 An Elegant Balancing Act: 

How Cells Maintain their Genetic 

Stability

Eberhard Fritz 

 

 

 

H. Lekscha; G.Bergner; E.Stöckl 

Diana Kirchhof 

49 60 72 

 

83 

Zhao-Qi Wang 

Swen Löhle 

 

 

 

Jürgen Sühnel 

 


52 64 74 

 

 

 

Peter Hemmerich 

 

Matthias 84 Platzer 

 

 

Jan Tuckermann 

 

 

Falk Weih 

 

 

55 66 

 

 

 

Heike Heuer 

77 

K.H. Gührs B. Schlott 

 

Matthias Görlach 

 


 

 

Christian Hoischen 

 

 

C. Calkhoven, H. Heuer, C. Kaether 

 

 

M. Than 

 

74 Steroid Hormones: 

 

 

Chairman: Wolfram Eberbach 

 

 

 

Peter Herrlich 

46 58 68 

Head of Administration: Daniele Barthel 

80 

Muttermal Fehlgebildetes 

Muttermal 

Radiales 

Wachstum 

Vertikales 

Wachstum 

HERRLICH LAB 

49 Metastatic Migration: 

A Disastrous Property of Cancer Cells 

Tumor & 

Metastasenbildung 

HEUER LAB 

52 Influential Messengers: 

How Thyroid Hormones Affect the Brain 

KAETHER LAB 

55 Misguided Proteins: 

Looking for the Causes of Alzheimer’s 

Disease 

MORRISON LAB 

58 How Switch Proteins are Regulated 

to Control Proliferation and Neural 

Function 

PLATzER LAB 

60 Genomes, Diseases and Ageing 

PLOUBIDOU LAB 

64 Virus-Induced Signal Transduction 

and Oncogenesis 

SCHILLING LAB 

66 Molecular Mechanisms of Huntington’s 

Disease and Therapeutic Approaches 

SüHNEL LAB 

68 From Information to Knowledge: 

New Databases and Analysis Tools 

THAN LAB 

72 From Structure to Function: 

How Proteins Work in the Body 

TUCKERMANN LAB 

 

 

Chairman: Piet Borst 

 

 

 

 

 

Benita Rost 

 

 

Regulators of Tissue Integrity, Metabolism 

and Inflammation 

WANG LAB 

77 Out of Balance – 

How Genomic Instability Promotes 

Diseases and Ageing 

WEIH LAB 

80 Vital Communication: The NF-κB Signal 

Transduction Pathway in the Immune 

System 

83 Organisation Chart 

84 Imprint 

3

4 Preface 

As of 2003, the former Institute for Molecular Biotech- 

nology (IMB) has been systematically reorganised into an 

institute devoted to research on ageing. In 2005 our new 

name put the seal on this process. We are proud to 

present the first national institute in Germany with an ex- 

plicit research focus on the mechanisms of ageing and 

age-related diseases. 

Why research on ageing? And why in Jena? In view of 

the increasing lifespan in industrialised nations, the 

theme is certainly timely. Equally important, however, is 

the current technological repertoire that enables us to 

study complex biological processes. What actually deter- 

mines ageing is still poorly understood. The field of age 

research today resembles that of cancer research some 30 

to 40 years ago, when environmental, genotoxic and ge- 

netic hypotheses existed side by side. Numerous ageing 

hypotheses (e.g. the “mitochondrial hypothesis on age- 

ing”, the “stem-cell hypothesis on ageing”, “genomic in- 

stability”, the “neuroendocrine hypothesis on ageing”) 

have found experimental support and co-exist without 

any proven, unifying theory. Likewise, age-related diseases 

are “multifactorial”, indicating that most of the mecha- 

nisms are yet unknown. 

Dear Readers, 

Several factors make Jena an ideal location for an in- 

stitute devoted to the study of ageing. The analysis of 

complex genetic mechanisms requires high-quality ge- 

nome analysis. In the past, the institute has contributed 

significantly to the sequencing of the human genome 

(2% of the human genome) and the genomes of other or- 

ganisms. We have further improved and upgraded the se- 

quencing technology that now enables us to rapidly de- 

tect genetic and epigenetic traits. Another unique feature 

of the FLI has been created by a research focus on ge- 

nomic instability, particularly on DNA repair, an area 

which in the past has been by and large neglected in Ger- 

many. This area is important for age research because hu- 

man syndromes caused by DNA repair deficiencies are 

characterised by premature ageing. Interestingly, DNA re- 

pair diseases go hand in hand with neurodegeneration. 

Neurological ageing is another focus of research at FLI. 

Although the neurosciences are well established at vari- 

ous national centres, our institute in Jena chose to con- 

centrate on specific issues in age-associated neural dis- 

ease investigated by experts newly recruited to the FLI. 

Animal models have become a major tool in neurodegen- 

eration research. At the same time, the Medical Faculty of 

the Friedrich Schiller University created a research focus 

on age-related diseases that complements our institute´s 

basic research. The increasing links with the Medical Fac- 

ulty will be profitable for both sides.

With the help of both the Federal government (BMBF) 

and Thuringian Ministry of Education and Cultural Affairs 

(TKM) major improvements for the infrastructure have 

been planned and are currently being implemented. The 

FLI is to be housed in a new building designed by the ar- 

chitect M. Mackenrodt (Archiscape, Berlin). This design 

was chosen by a high-ranking jury consisting of the archi- 

tects Otto Steidle, Benedikt Tonon, Arno Lederer and Tho- 

mas Bahr. Completion is planned for 2009/2010. Although 

the scientists recruited to Jena are of prime importance, 

the laboratory conditions in the new building will cer- 

tainly add to our institute’s international visibility and at- 

tractiveness. 

With this brochure we want to present the overall sci- 

entific concept and the individual projects of our research 

activities. Additional information on our institute is avail- 

able in our annual report “Facts and Figures 2006”. 


Scientific Director, 

Head of Institute 

5

6 Fritz Lipmann 

Fritz Lipmann: Biochemist, Nobel Prize Laureate 

and Pioneer of Age Research 

By choosing the name of Fritz Lipmann for our insti- 

tute, the FLI intends to honour an outstanding biochemist 

who contributed considerably to understanding the fun- 

damental factors involved in the ageing process. Fritz Lip- 

mann came from a Jewish family 

in Königsberg (now Kaliningrad). 

He received his chemical and 

medical education in Königs- 

berg, Munich and Berlin, and the 

Berlin-Dahlem research environ- 

ment influenced his first labora- 

tory work. After staying one year 

in New York, Fritz Lipmann 

moved to Copenhagen in 1932, 

and returned again to the United 

States in 1939, where he lived 

and worked in New York and 

Boston (1941 – 1957). In 1953 he 

was awarded the Nobel Prize for 

medicine or physiology for the 

discovery of coenzyme A, one of 

the most important factors in 

cellular metabolism. Fritz Lip- 

mann devoted much of his work 

to research on the energy me- 

tabolism of cells. In 1937 and 

1939 he presented a concept of 

Fritz Lipmann investigated the energy metabolism 

of the cells and discovered coenzyme A 

ATP production by mitochondria, a process known today 

as “oxidative phosphorylation”. ATP stands for adenosine 

triphosphate, the most important “energy currency” of 

the cell. In the 1940s Fritz Lipmann conducted research on 

the use of ATP for the regulation of pro- 

teins in the cell: A high-energy phosphate 

from ATP is transferred to a protein, 

thereby modulating its function. During 

this process, ATP is broken down into the 

lower energy form ADP (adenosine di- 

phosphate). 

In 2000 the Biographical Memoirs of 

the Royal Society had this to say about 

Lipmann’s achievements: “Fritz Lipmann 

was largely responsible for identifying 

and characterizing the connection be- 

tween metabolism and the energetics of 

living systems that makes life possible.” 

The current, still rudimentary knowledge 

on the connection between metabolism, 

life expectancy and reduced energy pro- 

duction by the mitochondria in ageing 

organs is based on Fritz Lipmann’s find- 

ings and has laid the foundations for age 

research at the cellular level. 

Together with Hans 

Krebs, Fritz Lipmann 

was awarded the Nobel 

prize in 1953.

Solving the Puzzle of Ageing 

Why do we age? This question is not new, it has preoc- 

cupied humankind for a long time. What is new is the de- 

mographic development over the last 100 years, which in- 

dicates that average life expectancy in the industrialised 

nations increases by 3 months a year or 6 hours a day. The 

resulting increase in the ageing population alarms politi- 

cians and prompts the biomedical community to increase 

its efforts to understand the ageing process and to ex- 

plore how we can offset age-related diseases such as 

Alzheimer’s and cancer. The cost to society that is involved 

in this demographic development is by no means the only 

issue to be addressed, indeed it is not clear whether the 

health costs now rocketing around 

age 60 will increase considerably 

with the number of centenarians. 

Quite apart from the costs, the in- 

creased number of possibly un- 

healthy people is a considerable 

challenge for humanity. The desire 

to grow older in as healthy a condi- 

tion as possible is a profound 

concern. One of the most pressing 

challenges involved in this research 

is that of contributing to healthy 

ageing. 

Healthy ageing 

Research on ageing is complex 

indeed. Numerous unanswered 

Life expectancy of people living in Europe 

has been rising steadily over the past 100 years. 

Exception: the war years 

The Puzzle of Ageing 

questions need to be addressed: How much do external 

and internal causes contribute to ageing? To which ex- 

tent is the individual ageing process predetermined by 

the genetic constitution? Do cellular functions simply de- 

teriorate in the older years of an organism, or is the 

lifespan predetermined during embryogenesis? Although 

currently disfavoured, not even the question of whether 

there is such a thing as a genetic clock dictating lifespan 

has been answered unequivocally. 

The prime and ultimate goal of biomedical age re- 

search is to break the link between ageing and disease. To 

achieve this goal, it is important to 

explore the mechanistic determi- 

nants of ageing and the links with 

disease, for instance to identify oper- 

ative differences between individu- 

als who live to an old age without 

ailments and others who suffer from 

diseases. The Fritz Lipmann Institute 

hosts a number of research activities 

that address this overall goal. But to 

be successful, we need to focus on 

specific selected topics approached 

from a variety of different perspec- 

tives. The next section describes 

how the FLI research laboratories are 

thematically organised to address 

such topics. 

7

8 Research Concept 

Heads of laboratories at FLI 

Many Roads – One Goal 

Within the overall field of age research, the scientists 

at the Fritz Lipmann Institute are involved in two major 

programmes: “Mechanisms of Ageing and Senescence” 

and “Age-Associated Diseases” (see diagram). The penta- 

gons for each programme indicate the subtopics covered 

by the research groups. The whole research area, with its 

formally divided programmes, is characterised by an enor- 

mous degree of scientific and methodological overlap, 

both between and within the programmes and their sub- 

topics. 

The increasing scientific coherence of the research lab- 

oratories of the FLI over the last three years has brought 

with it a high degree of collaboration and synergy in their 

efforts to deal with related issues. As the illustration indi- 

cates, most FLI laboratories contribute to a variety of dif- 

ferent subtopics (pentagons), by investigating similar 

questions using individual experimental expertises and 

approaches. The laboratories will be presented separately 

in this brochure however the links between the labs will 

also be indicated. 

Scientific coherence in the institute is also reflected by 

a number of regular lab meetings, journal clubs and dis- 

cussion rounds shared between different laboratories. 

The „Metabolic Club“ (Bauer, Calkhoven, Heuer, Tucker- 

mann labs) and „Genomic Instability“ lab meeting (Greu- 

lich, Herrlich, Wang labs) may serve as prominent exam- 

ples for such joint regular meetings of several laboratories 

on collective research topics. The fact that many labs 

share a high degree of methodological overlap addition- 

ally promotes coherence of the research groups.

9

10 Research Concept 

Mechanisms of ageing and 

senescence 

Laboratories working on the “Mechanisms of 

Ageing and Senescence” encompass research on 

lifespan in a new model organism, on the identifi- 

cation of the determinants of healthy human age- 

ing and on selected aspects of cellular senescence 

under the heading of “Longevity”. A significant 

role in both organismic ageing and cellular senes- 

cence is probably played by “Destabilisation of 

the genome”, which accordingly represents a 

major focus in the work done at the FLI: Several 

FLI laboratories concentrate on aspects of DNA 

replication, DNA repair, chromosome segrega- 

tion and telomere structure and maintenance. 

Errors in these processes cause premature ageing 

in cells and organisms and are key factors in the 

development of age-associated diseases. In addi- 

tion, the enormous expertise in genome analysis 

available at the FLI is being used to identify age- 

dependent DNA methylation of the human ge- 

nome, changes in gene copy number and in the 

pattern of splicing. 

Age-associated diseases 

The second major focus at the FLI is research on age- 

associated diseases, notably selected aspects of neurode- 

generation and cancer. In addition, specific issues are ad- 

dressed in the area of the metabolic syndrome, including 

associated conditions of hormonal dysregulation and 

atherosclerosis. Within the subtopic “Impaired tissue ho- 

moestasis”, osteoporosis and chronic inflammation are 

studied, among others. Since embryonic processes are 

partly recapitulated in tissue regeneration, developmen- 

tally active genes related to kidney and gonadal disorders 

are studied in a zebrafish model. In the subtopic “Geno- 

mic variability” the FLI is identifying disease-associated 

polymorphisms through genome analysis. Almost all FLI 

activities on age-associated diseases rely on the use of an- 

imal models, currently mice and fish. 

Support technologies 

As one might expect, a whole range of methods and 

technologies is needed to explore the processes involved 

in ageing and disease, from the study of single molecules 

and cells in culture to animal models and the study of 

families whose members display longevity. Using such 

different mutually supportive methodologies and experi- 

mental approaches to answering basic questions about 

molecular functions is one of the strengths of the Fritz 

Lipmann Institute. 

The methodical synergies at FLI range from structural 

biology and protein biochemistry to cell biology and from 

the use of cell culture models to animal models for typical 

human age-associated diseases. We also analyse bio- 

probes from selected patient cohorts, while old-aged indi- 

viduals are used for genetic and molecular analysis of age- 

ing and pathogenic mechanisms in humans.

Research environment 

As in other areas, we expect that 

mechanistic knowledge will rapidly ac- 

cumulate in the area of age research. It 

takes, however, time before applications 

reach the patients or the ageing individ- 

ual. To catalyse the transfer of research 

results to clinical studies as quickly as 

possible is therefore within FLI’s atten- 

tion. In order to promote the interac- 

tions between basic scientists and clinicians, we are cur- 

rently setting up a Leibniz Research School for Clinician 

Scientists. The additional benefit of this school is to per- 

mit clinicians to develop their own research in a stimula- 

tory environment and with reduced clinical duties. 

The research interests of the scientists working at the 

Fritz Lipmann Institute are closely linked with those of the 

Friedrich Schiller University (FSU) in Jena, other research 

institutes on the local Beutenberg campus and many 

other cooperation partners at national and international 

institutions. Several research group leaders at the Fritz 

Lipmann Institute have professorships at the Friedrich 

Schiller University and contribute substantially to teach- 

ing at the FSU. The recently established Graduate School 

of the FLI (the Leibniz Graduate School on Ageing and 

Age-Related Diseases, LGSA) is designed to ensure future 

continuity in age research. 

More information 

selected projects are highlighted. 

In this brochure the group leaders 

and scientists of the Fritz Lipmann Insti- 

tute introduce their research projects 

and scientific achievements to the gen- 

eral international public for the first 

time. Each research laboratory is pre- 

sented as an individual entity, pinpoint- 

ing its specific contributions to the over- 

all research design. In addition, a few 

For more information, please visit our website 

www.fli-leibniz.de. An overview of the recent scientific 

output and the structure of the FLI is presented in our 

annual report “Facts and Figures 2006”, available on our 

website or in hard copy form on request. 

11

12 Selected Topics: Tumour Biology 

How Fatal Growth Stimuli Are Blocked 

Normal cells in the developing organism know exactly when to stop proliferating. When an 

organ is formed and the space filled, normal cells no longer react to growth stimuli. Tumour 

cells, however, have forfeited this property. They go on growing, even pushing other tissues aside in 

the process. The laboratory headed by Helen Morrison explores the mechanisms involved in growth 

control. She describes a chain of reactions involving a protein cascade telling cells that they have 

made contact with their neighbours (cell-cell contact). This is a novel regulatory step in cellular 

signalling that goes off the rails in tumour cells. 

Neurofibromatosis type II (NF2) is a rare inherited dis- 

ease that affects 1 in 40,000 individuals. NF2 patients de- 

velop a variety of tumours in the nervous system, the 

most notable of which are vestibular schwannomas, tu- 

mours associated with hearing nerves. NF2 patients carry 

defects in a gene specifying the production of a protein 

called merlin. Defective merlin (or its absence) causes ab- 

normal and uncontrolled cell proliferation leading to the 

formation of tumours. In most, if not all, normal cells of 

the organism, merlin controls cell multiplication (and 

hence is called a tumour suppressor protein). Merlin ex- 

ists, however, in two states: an active state, in which it in- 

hibits cell proliferation, and an inactive state in which it 

permits cell proliferation. Accordingly, merlin itself is regu- 

lated and only becomes active under certain conditions. 

Switching between the active and inactive states appears 

to be controlled by the absence or presence of a phos- 

phate group on the merlin molecule at the location called 

ser518. These are very basic mechanisms. The transfer of a 

high-energy phosphate from ATP to the proteins is 

known as phosphorylation (see the section on Fritz 

Lipmann), while removal of the phosphate is known as 

dephosphorylation. 

Tight control of merlin activity 

Merlin is inactive when the phosphate group is at- 

tached to ser518 and active when it is removed. Merlin is 

only dephosphorylated at the ser518 position following 

cell-cell contact and only such activated merlin stops cel- 

lular proliferation. The Morrison laboratory has explored 

how merlin manages to halt proliferation and how the 

dephosphorylation of merlin is achieved. Put briefly, the 

result was the discovery of a reaction cascade involving 

several stages. Merlin interferes with the activation of a 

well-known proliferation-promoting protein called Ras. 

Like merlin, Ras behaves as a switch which in the “on” po- 

sition predominantly induces cells to multiply. Lack of

Molecular signal cascades influence a cell’s proliferation: 

after an extracellular growth factor is bound to its receptor 

this message is further transferred via several intracellular 

proteins (Grb2, SOS, Ras, Raf, MEK, ERK). The transient nature 

of this signal is characterised by reversible changes of the 

proteins involved, i.e. modification of amino acids (pY) or 

exchange of co-factors (Ras-GDP & Ras-GTP). The cell will 

respond to this signal and adjust its behaviour accordingly, 

e.g. divide. 

P Y 

Growth 

factor 

receptor 

Y P 

Growth 

factor 

Grb2 

Sos 

Ras 

GDP 

Ras 

GTP 

Cell membrane 

Raf 

MEK 

ERK 

Growth 

merlin leads to a hyperactive form of Ras resulting in un- 

controlled cell proliferation and thus ultimately contribut- 

ing to the formation of cancer. 

Merlin is dephosphorylated in response to a stimulus 

from outside the cell that is provided by cell-cell contact. 

A significant step towards understanding this process was 

recently achieved by our discovery of the enzyme respon- 

sible for removing the phosphate from merlin. This en- 

zyme is a phosphatase known as the myosin phosphatase 

(MYPT-1–PP1δ) previously known to be involved in con- 

tractility, a process important for cell shape and motility. 

The enzyme specifically selects merlin from among many 

other proteins. The specificity is mediated by one of the 

sub-units of the phosphatase, the MYPT sub-unit. MYPT 

makes the contact and guides the catalytic sub-unit PP1δ 

to its target (merlin). Not surprisingly, a regulatory reac- 

tion of this importance is subject to further control. For 

example, the cells can produce an inhibitor called CPI-17, 

and both CPI-17 and the phosphatase itself are again regu- 

lated by phosphorylation. We now hope to identify other 

steps in the cascade mediating the activation of the tu- 

mour suppressor protein merlin in response to cell-cell 

contact. 

Our results extend the current model of cellular growth 

control by three essential findings: the growth factor is 

dependent on a co-receptor (mostly adhesion proteins). This 

co-receptor functions as an anchor on the intracellular face by 

binding to the protein ezrin, which connects to the cellular 

skeleton (actin filaments). Ezrin in turn associates with and 

activates the signalling component SOS thereby directly 

participating in the signal relay. 

P Y 

Growth 

factor 

Growth 

factor 

receptor 

Y P 

Grb2 

Adhesion 

receptor 

SOS 

Ezrin 

Ras 

GDP 

Actin filaments 

Extracellular 

matrix 

Cell membrane 

Ras 

GTP 

Dysregulation of merlin promotes 

tumourigenesis 

Raf 

MEK 

ERK 

Proliferation... 

Tumour cells arise when merlin is defective (as in neu- 

rofibromatosis type 2). Can tumours form when merlin is 

normal but cannot be activated by dephosphorylation? 

We have indeed found evidence for this possibility. Under 

certain conditions, elevated expression of the inhibitor 

CPI-17 causes cultured cells to become tumour-like. Re- 

cently we also discovered several human cancers that 

carry high levels of CPI-17. In these cells, merlin is inactive 

and the growth stimuli constantly push Ras into the “on” 

position. 

While merlin inactivation is a major factor keeping Ras 

in the “on” position, we also wanted to know whether this 

absence of an “off” switch for merlin was sufficient in it- 

self. Our work on ezrin (see Morrison lab, page 58) indi- 

cates that merlin and ezrin (which is structurally related 

to merlin) compete for the same interaction sites on the 

plasma membrane. Ezrin promotes Ras-dependent signal- 

ling and subsequent cellular proliferation by binding Ras 

and localising it to a specific Ras-activating enzyme called 

SOS. In addition, ezrin can interact with SOS and speed up 

the catalytic activity of this enzyme. Merlin, however, 

does the opposite! While active dephosphorylated merlin 

can localise to the same sites in the plasma membrane as 

13

14 Selected Topics: Tumour Biology 

A single cell layer of contact inhibited cells is visible in the 

electron microscope. Experimental activation of the oncogene 

CPI-17 transforms cells characterised by loss of cell to cell 

contacts, change of shape, and enhanced proliferation. 

The graphic depicts the mode of action of CPI-17: it inhibits the 

myosin phosphatase MYPT-PP1δ. This phosphatase is a crucial 

activator of the tumour suppressing protein merlin. 

 

 

 

 

ezrin, merlin cannot interact with SOS and activate it. 

How do ezrin proteins know that they need to stop func- 

tioning? Both merlin and ezrin are regulated by the same 

mechanism. Ezrin is activated by the transfer of phos- 

phate, whereas phosphorylated merlin is inactive. Cell-cell 

contact triggers MYPT-PP1δ-dependent dephosphoryla- 

tion, which removes phosphates from both types of pro- 

tein, subsequently activating merlin and at the same time 

inactivating ezrin. This enables merlin to interfere with 

the action of the ezrin proteins and to block the essential 

signalling events promoting growth. When CPI-17 is ab- 

normally expressed, the phosphatase MYPT-PP1δ will be 

inhibited, thus causing the cells not only to lose merlin 

but also to acquire the tumour-promoting activity of 

ezrin. Eventually we hope to discover tools that can inter- 

fere with the action of CPI-17. 

Authors: Helen Morrison, Tobias Sperka and Peter Herrlich 

Phone: 0049-3641-656139 

E-mail: helen@fli-leibniz.de 

Original publication: 

Tumorigenic transformation by CPI-17 through inhibition of a 

merlin phosphatase, 

Nature 442, 576-579. 

CPI-17 therefore arrests merlin in the inactive state allowing the 

cell to proliferate. 

δ 

 

 

 

 

 

 

 

 

 

 

 

 

 

Helen Morrison and the Thuringian Minister of 

education and cultural affairs Jens Goebel during the 

awards ceremony for the Thuringian Research Award

Dictyostelium discoideum, a small model for 

big questions: Why and how did 

multicellular organisms like humans evolve? 

Dictyostelium discoideum is an amoeba that 

remains unicellullar… 

Huddling Together for Safety: 

How Cells Cooperate 

What is it that enables single cells to aggregate into a 

multicellular organism? Though we still cannot give a 

complete answer to this question we have made consider- 

able progress in that direction. In 2005 a Dictyostelium 

Genome Analysis Consortium assembling scientists from 

the United States, the United Kingdom, Japan and Ger- 

many published a description and initial analysis of the 

genome of the amoeba Dictyostelium discoideum in the 

journal Nature. 

What makes an amoeba so interesting? Its specific life- 

form makes Dictyostelium what it has been for decades al- 

ready: a model system for scientists attempting to find 

out how organisms develop, how cells transmit messages 

(signal transduction) and the role played by the cell skele- 

ton (cytoskeleton). 

Social when need be 

The most striking feature of these amoebas is their veg- 

etative life-cycle. It is characterised as follows: When ambi- 

Selected Topics: Genome Analysis 

Dictyostelium discoideum is an amoeba that remains unicellular as long as conditions are 

favourable. When conditions deteriorate, these individual cells congregate to form a multicellular 

organism, which then develops a long, thin stalk and a fruiting body dispersing spores. 

How do these single cells communicate? What enables them to join forces? How do they specialise? 

Gernot Glöckner has the answers to these intriguing questions and indicates their relevance for 

human ageing. 

ent conditions become unfavourable, single autonomous 

cells aggregate into a multicellular fruiting body after un- 

dergoing several well-defined interim stages. Only a part 

of these cells will develop into viable forms and survive 

the aggregation process. In no other branch of the tree of 

life do individual and essentially autonomous cells cooper- 

ate in this way. They assure the survival of the species by 

sacrificing themselves. This astonishing altruism can only 

function if the cells communicate and single cells special- 

ise into cell types performing specific tasks (cell differenti- 

ation). Incidentally, this conditional multicellularity of the 

amoeba is achieved largely with the same set of genes 

that permanently multicellular systems draw upon. 

Another remarkable feature of Dictyostelium is the 

amoeboid motility of the cells, which is assured by the 

components of the cytoskeleton. The components and dy- 

namics of the cytoskeleton are readily comparable to the 

cytoskeleton of cells moving freely in multicellular organ- 

isms, for example the macrophages, scavenger cells of the 

immune system. 

15

16 Selected Topics: Genome Analysis 

The amoeba D. discoideum has 6 chromosomes and around 12,000 genes. The adjacent 

picture shows the fruiting body, which is built as end-point of the vegetative multicellular 

life cycle of the amoeba. The lighter cells in this picture are destined to die, whereas the 

dark cells will survive and develop to spores. 

Happy Mapping 

Deciphering the genome of Dictyostelium discoideum 

was a stiff challenge. First, the percentage of complex 

repetitive elements (10%) is high for such a small genome 

(34 Mb). Another complicating factor was that the pro- 

portion of the nucleotides adenin and thymin is so high 

(78%) that bacterial subclones can only be kept stable up 

to a size of five kilobase (in genetics a kilobase, kb, is a 

unit used to measure lengths of DNA and corresponds to 

1,000 nucleotides). Accordingly, sequencing with the as- 

sistance of large bacterial clones was impossible. To en- 

sure that the entire genome was represented it was nec- 

essary to establish an accurate map of it. To this end, a 

second map charted with the “Happy Mapping” method 

was integrated into an existing rough genetic map to 

achieve an average landmark density of 1/60 kb in the ge- 

nome. A map of this accuracy was essential to identify the 

entire chromosomal structure. In the process a striking 

feature became apparent. At its end (terminus), every 

chromosome displays a region consisting largely of clus- 

ters of special retro-elements. Our conjecture is that these 

regions perform the function of centromeres to which the 

spindle fibres attach during cell division in order to segre- 

gate the chromosomes after duplication. Another feature 

common to all the chromosomes is that their termini end 

in sequences deriving from the so-called rDNA palin- 

drome, which figures at various points in the cell nucleus 

and carries the information for the ribosomal RNAs. The 

transition from chromosomal to palindrome sequence 

does not appear to be defined, as the attachment point 

differs for each chromosome terminus. Thus one might 

regard the chromosomes as sequences embedded in an 

acentric palindrome. As the chromosomal termini do not 

display any differences from the rDNA palindrome, they 

may conceivably be repeatedly renewed from this reser- 

voir. In other words, Dictyostelium has found a way of us- 

ing the same components over and over again to form 

protective caps for its chromosomes. 

Genes of an amoeba 

Dictyostelium’s small genome contains over 12,000 

genes, a similar number to the genomes of “genuine” 

metazoa (multicellular organisms) like the fruit-fly Dro- 

sophila. Apart from a small number of genes probably 

restricted to this evolutionary line, most of the Dictyostel- 

ium genes are similar to those of other organisms. Re- 

markable are a number of genes and gene families for- 

merly thought to be the “invention” of metazoa only. 

In relation to the number of genes, we found consider- 

ably fewer transcription factors than is customary for 

other species. Transcription factor is the term used for 

proteins that can switch genes on or off. So far, just under 

100 transcription factors have been identified in Dictyos- 

telium, fewer than in yeasts. Transcription factors with the 

so-called “basic loop-helix-loop domain” are totally ab- 

sent. This motif has been identified in all other evolution- 

ary lines so far.

This relative absence of transcription factors in Dicty- 

ostelium may have to do with the use of more highly inte- 

grated regulation mechanisms. It is however more likely 

that most transcription factors are specifically adapted to 

the requirements of the genome and have yet to be dis- 

covered because they have little or no similarity to known 

counterparts in other organisms. Further investigation is 

necessary to clarify this issue and to use a systems biology 

approach to describe the regulatory network of these or- 

ganisms. 

What we have so far is the entire sequence, i.e. the 

complete order of all bases constituting the genome of 

Dictyostelium. This makes it possible for us to analyse the 

functions of gene families. As the functions of gene prod- 

ucts (proteins) of a gene family frequently overlap, we use 

“knock-out” mutants in which individual groups of genes 

are systematically switched off. In this way we can include 

the entire genetic background and describe the cause and 

effect relations. 

Another field of research opened up by our knowledge 

of the entire genome sequence of Dictyostelium is the 

comparison of the genomes of different organisms (com- 

parative genomics). Various social amoebas have been de- 

scribed morphologically and are present in strain collec- 

tions. Other members of the same evolutionary branch 

are organisms as various as Entamoeba histolytica or 

Physarum polycephalum. We now have the prospect of 

embarking on in-depth study of the evolution of this fas- 

cinating group of organisms. 

Protective caps for chromosomes 

Telomeres form the termini of linear chromosomes, 

closing them off like protective caps. To ensure that no 

genetic material gets lost they have to remain intact 

throughout the entire life of the cell. If the machinery 

maintaining the telomeres is damaged, the cell will die. 

This factor may also be involved in the ageing of complex 

organisms like the roundworm Caenorhabditis elegans, 

the fruit fly Drosophila or humans. The structure of the 

telomeres of Dicytostelium discoideum differs fundamen- 

tally from the telomere structure of other model organ- 

isms used in age research and also from that of the hu- 

man organism. In a project entitled “From Comparative to 

Functional Genomics of Social Amoeba” funded by the 

German Research Foundation (DFG) we have set out to 

elucidate the mechanisms ensuring telomere preservation 

in Dictyostelium discoideum. We believe that this project 

will make a substantial contribution to our understanding 

of ageing. 

Author: Gernot Glöckner 

Phone: 0049-3641-656440 

E-mail: gernot@fli-leibiniz.de 


The genome of the social amoeba Dictyostelium discoideum. 

Nature 435, 43-57 

Contrary to the situation in other 

organisms the telomeres of 

Dictyostelium are not composed 

of simple repeated sequences. 

What mechanism do the 

amoebae use to maintain their 

chromosome ends? And can this 

mechanism be exploited to better 

understand ageing, even in 

humans? These are two burning 

questions of the group of Gernot 

Glöckner. The figure shows the 

detailed telomere structure of 

D. discoideum. 

17

18 Selected Topics: Biophysics 

Virus-Induced Cancer: 

The Structural Basis of Viral Cancerogenesis 

Certain viruses, the so-called papilloma viruses, may cause cancer in humans. The molecular 

details of how the virus manages to transform a healthy cell into a cancer cell are still enigmatic. 

Matthias Görlach explains how a virus removes the “brakes” in infected cells to drive them into 

uncontrolled proliferation. The laboratory has succeeded in determining a protein structure of the 

virus that appears to be an appropriate target for drugs intercepting the molecular pathways the 

virus draws upon to trigger cancer. 

Human papilloma viruses infect the basal layers of epi- 

thelia, such as skin or mucosa, thereby causing a number 

of medical conditions ranging from harmless warts to 

cancer. Accordingly, the papilloma viruses are categorised 

into different types posing low or high risk of causing can- 

cer (LR = low-risk; HR = high-risk). 

The virus types 16, 18 and 45 belong to the high-risk 

group. They cause cervical carcinoma, the second most 

frequent form of cancer affecting women worldwide. 

However, we still have only an imperfect understanding 

of how exactly an infection with these virus types ulti- 

mately leads to the development of cancer. It is known 

that, together with cellular factors, certain proteins of the 

virus, the so-called oncoproteins E6 and E7, contribute de- 

cisively to the transformation of cells in cervical mucosa. 

The oncoprotein E7 operates by interacting with cellular 

proteins, among them pRb and p21CIP. These two proteins 

are involved in regulating the processes of the cell cycle. 

Our aim is to elucidate the structure of E7 proteins derived 

from low-risk and high-risk papilloma virus types. In addi- 

tion, we intend to investigate how this viral protein inter- 

acts with cellular proteins, thus shedding light on the 

structural basis of viral cancerogenesis. Once the modus 

operandi of the E7 oncoprotein is understood, i.e. the way 

it interacts with cellular proteins, it may be possible to 

find substances that can intercept such interactions. This 

in its turn could pave the way for the development of 

medical drugs preventing progression of pre-cancerous 

conditions (so-called pre-malignant lesions) into full- 

blown tumours.

How the virus subverts cellular growth 

regulation 

The oncoprotein E7 comprises approx. 100 amino acids, 

the building blocks of proteins. E7 contains three regions 

CR1, CR2 and CR3, which are conserved among the differ- 

ent papillomavirus types (CR = conserved regions). Of 

those, CR1 is unique and found only in E7, while CR2 is sim- 

ilar to equivalent regions of the E1A protein from adenovi- 

ruses and the large T antigen of SV40 viruses. The CR3 re- 

gion of E7 contains two CxxC motifs, which are separated 

by 29 amino acids and which co-or- 

dinate a zinc ion, thereby stabilis- 

ing the structure of E7. 

The CR2 region of the on- 

coprotein E7 contains a 

strictly conserved sequence 

motif (LxCxE) that contrib- 

utes decisively to the interac- 

tion of E7 with a cellular protein, 

the tumour suppressor pRb. Binding 

of pRb by E7 and subsequent E7 CR1 de- 

pendent pRb degradation by the cellular proteasome lead 

to a release of pRb-bound transcription factors of the E2F 

family. Transcription factors are proteins that switch genes 

on or off. Here, the transcription factors released initiate 

the read-out of genes that are necessary for the entry of 

cells into the S-phase of the cell cycle. The S-phase is 

mainly characterised, among other things, by replication 

(duplication) of the cellular hereditary substance DNA. 

The human papilloma virus 

(here HPV 16) causes cervical carcinoma, 

the second most frequent tumour in women 

worldwide. 

The CR3 region of oncoprotein E7 mediates contact 

with a number of cellular regulatory proteins, including 

p21CIP, the NURD histone deacetylase complex, BRCA1 and 

the insulin-like growth factor binding protein IGFBP3. 

Binding to E7 by the cyclin-dependent kinase inhibitor 

(CDKI) p21CIP abrogates the inhibition of cyclin-dependent 

kinases. This interaction also diminishes the inhibition of 

PCNA-dependent DNA replication as the binding sites for 

E7 and PCNA in the C-terminal region of p21CIP overlap. 

In short, the binding of the viral oncoprotein E7 

to the cellular regulatory proteins, e.g. 

pRb and p21CIP, leads to a re- 

lease of “molecular brakes”. 

In a healthy, non-infected 

state these brakes pre- 

vent cell division and 

proliferation. 

Portrait of an 

oncogenic protein 

In order to elucidate the structural 

basis of this virus-induced dysregulation of the 

“molecular brakes”, we are investigating the structure and 

the interactions of the viral oncoprotein E7 from a number 

of papilloma virus types (both LR and HR types). As a first 

step, the full-length form and the CR3 region of E7 from 

HR–HPV 45 were produced in bacteria in the presence of 

the stable isotopes 13C and 15N, purified and prepared for 

structural analysis by NMR spectroscopy. A comparison of 

19

20 Selected Topics: Biophysics 

[1H,15N] HSQC – “fingerprint” – spectra of the two E7 con- 

structs revealed that the N–terminal part (CR1 and CR2) of 

E7 is mainly unstructured, as has been observed in other 

regulatory proteins in the absence of their specific protein 

ligands. By contrast, the C-terminal CR3 region adopts a 

defined spatial structure, which in turn depends upon the 

presence of zinc ions. Complete structural analysis re- 

vealed that CR3 assembles into homodimers. Each mono- 

mer adopts a β1β2α1β3α2 topology and the stabilising zinc 

ion is co-ordinated by the four cysteine residues of the 

two CxxC motifs. This topology represents a novel pro- 

tein-folding motif, which is not found in other zinc-bind- 

ing cellular proteins. Relaxation measurements and mo- 

lecular dynamics simulations show that the β3 strand is 

formed as a consequence of dimerisation and stabilised 

by hydrogen bonding with the β2 strand of the other 

monomer. Accordingly, it is only stable in the dimer. The 

hydrophobic core of each monomer is comparatively small 

and dimerisation occurs via exposed hydrophobic residues 

of the individual monomer cores. As a result, a larger com- 

bined and contiguous hydrophobic core forms, which very 

likely contributes significantly to stabilising the CR3 struc- 

ture. 

NMR spectra of the viral protein E7 

constituting the basis for determination of its 

three-dimensional structure. 

The E7:p21CIP interaction was characterised by means 

of a series of titration experiments. Increasing amounts of 

a peptide representing the C-terminus of p21CIP were 

added to the E7–CR3 and the perturbation of the chemical 

shift of CR3 amide resonances induced by the binding of 

the p21CIP C-terminus was observed via NMR spectros- 

copy. This enabled us to “map” the binding site of p21CIP 

The NMR structure reveals surface properties 

important for contacts between the viral protein E7 

and cellular proteins. 

to a shallow groove between α1 and β2 of the CR3 of E7. 

Interestingly, this part of CR3 partially overlaps with one 

binding site of pRb on E7. Hence this region of the CR3 sur- 

face constitutes an initial target structure for the develop- 

ment of substances that may impede the binding of 

p21CIP and pRb to E7 and thus interfere with the patho- 

genic “modus operandi” of oncoprotein E7. 

This project profits from internal collaborations with 

the labs of A. Ploubidou and H. Morrison, who study the 

role of the cytoskeleton in oncogenic progression and sig- 

nalling pathways in tumour cells, respectively. 

Author: Matthias Görlach 

Phone: 0049-3641-656220 

E-mail: mago@fli-leibniz.de 


Solution structure of the partially folded high-risk human 

papilloma virus 45 oncoprotein E7 

Oncogene 25, 5953-5959.

What the Turquoise Killifish 

Can Tell Us about Ageing 

The turquoise killifish Nothobranchius furzeri is at 

home in East Zimbabwe and Mozambique, where it lives 

in seasonal waters such as those existing in the rain pe- 

riod only. To the best of our knowledge, fish of this spe- 

cies are among the vertebrates with the shortest lifespan. 

They have to make optimal use of the “season” in their 

native habitats, reach sexual maturity after only a few 

weeks, mate, lay their eggs and die before the next dry 

period. During the dry period the eggs laid in the muddy 

soil remain in a state of arrested development until the 

next monsoon, when they hatch, thus assuring the sur- 

vival of the next generation. 

Even under optimal conditions fish of this kind kept in 

the laboratory live only a few months. In addition, differ- 

ent populations of Nothobranchius furzeri show differ- 

ences in lifespan depending on the characteristic of their 

local habitat. Since these differences are genetic, this 

makes it an ideal model for identifying genes which con- 

trol longevity and ageing processes in natural populations 

like humans are. 

Selected Topics: Genetics of Ageing 

The African turquoise killifish Nothobranchius furzeri has an extremely brief lifespan of only few 

months. This model system can be used to test interventions for healthy ageing which are 

eventually of relevance for humans and to identify the genes controlling ageing rates in natural 

populations. The laboratories headed by Alessandro Cellerino, Christoph Englert and Matthias 

Platzer are using complementary approaches to tackle these two issues. 

Interventions for healthy ageing 

The identification of molecules able to prevent age- 

related diseases is a formidable challenge for our society. 

From the study of yeasts, fruit flies and nematode worms 

we know that resveratrol, a substance found in grape 

skins and in red wine, prolongs lifespan in these simple 

models. Whether this natural substance can also have a 

life-prolonging effect on vertebrates and – more impor- 

tantly – prevent age-related diseases was unclear. One 

reason for this is that life-long pharmacological experi- 

ments in rodents – the most widely used lab vertebrate – 

take years to be completed. Now, however, observations 

of Alessandro Cellerino’s lab obtained in the short-lived 

vertebrate Nothobranchius furzeri have indicated that 

resveratrol can indeed prolong lifespan and counteract 

age-related illnesses. 

The natural substance, resveratrol, was added at 

different concentrations to the fish-food and caused 

significant life-extension. More importantly, resveratrol- 

treated fish were physically fit, fertile and did not show 

21

22 Selected Topics: Genetics of Ageing 

age-dependent brain degeneration and senile cognitive 

impairment. Similar effects can be achieved by lowering 

the water temperature in the aquarium from 25 to 22 de- 

grees Celsius. 

An embryo of Nothobranchius furzeri shortly before 

hatching: Eye and tail are clearly visible. Embryos of 

the fish develop in the egg over several weeks, thereby 

passing through two to three dormant stages. 

This paradigm can now be extended to other classes 

of substances to identify drugs which can 

be suitable to prevent the occurrence of 

age-related diseases in humans. 

First steps towards 

a genome project 

We have next to no molec- 

ular-genetic data for Notho- 

branchius furzeri. Accordingly, 

we initiated a “Notho- 

branchius furzeri genome 

project” to establish the turquoise 

killifish as a new model for age 

research. The lab headed by 

Matthias Platzer had already been involved in 

the deciphering of the human genome and 

that of other organisms. In the long-term perspective, the 

Nothobranchius furzeri genome project will represent the 

basis for all further molecular, cell-biological and whole- 

organism investigations. 

In the first stage, thousands of random sequences 

were determined in the Nothobranchius genome, indicat- 

ing how large the fish’s genome is and how many se- 

quences (chemical building blocks) recur regularly. 

Brain structure of the fish 

Nothobranchius furzeri grows to a maximum of 

seven centimetres in length – and some of its species 

have a lifespan of only three months. 

Identification of genes controlling longevity 

in natural populations 

Our knowledge of the genetic control of ageing comes 

from studies where specific genes were artificially mu- 

tated in genetically homogeneous laboratory animals. 

Very little is known concerning the control of lon- 

gevity in genetically variable natural popula- 

tions. 

The lab headed by Alessandro 

Cellerino has discovered that different pop- 

ulations of Nothobranchius furzeri originating 

from regions with shorter or longer dura- 

tion of the monsoon show remarkable 

differences in longevity and timing of 

expression of age-related pathologies. 

These populations interbreed freely 

and produce offspring of intermedi- 

ate lifespan. These results set the ba- 

sis to identify the genes responsible for 

these differences. 

The lab of Matthias Platzer has identified 

hundreds of so-called genomic landmarks which differ 

between populations. Combining functional and genomic 

approaches, the analysis of hybrids of short-lived and 

long-lived population can now allow the identification of 

the chromosome regions which are responsible for differ- 

ences in longevity and age-related pathologies. In the sec- 

ond hybrid generation, a segregation takes places so that 

both shorter-lived and longer-lived individuals are gener- 

ated. The lifespan and occurrence of age-related disease 

in each hybrid fish is then recorded and correlated with 

inheritance of specific genomic landmarks.

Gills (green) of Nothobranchius in a microscopic image. 

Work on this issue will take place at the level of the 

entire genome but will also be complemented by compar- 

ative analysis of “candidate” genes which influence 

lifespan in other model systems. 

Can gene transfer prolong life? 

For some years now, the laboratory headed by Chris- 

toph Englert has been studying zebrafish to find out how 

genes control organ formation. The functions of individ- 

ual genes can be detected by switching off certain genes 

or introducing additional ones. In future we intend to ex- 

tend these experiments to Nothobranchius furzeri. The 

crucial issue here is the molecular basis of the ageing 

process and the way it determines lifespan. At present we 

are isolating a number of candidate genes from the ge- 

nome of Nothobranchius furzeri, genes known from ex- 

periments on other species to be an operative factor in 

ageing. This in its turn is the prerequisite for systematic 

manipulation of age-associated genes. Above all, it will be 

interesting to introduce genes from short-lived Notho- 

branchius subspecies into those with a longer lifespan 

and vice versa. We may confidently expect these experi- 

ments to tell us something about the way humans age. 

Research expedition to Mozambique: 

Nothobranchius furzeri has been discovered in its 

natural habitat for the first time in 35 years. 

Authors: Alessandro Cellerino, Christoph Englert, 

Matthias Platzer 

Phone: 0049-3641-656336 

E-mail: acellerino@fli-leibniz.de, 

cenglert@fli-leibniz.de, 

mplatzer@fli-leibniz.de 

23

24 Selected Topics: Bioinformatics 

Analysis and Interpretation of Complex Data 

T he Jena Centre for Bioinformatics supports and links bioinformatics research in Jena. Bioinformatic 

analysis and interpretation is also of importance for the exceptionally challenging task of 

research into ageing. 

Does bioinformatics have anything to do with ageing? 

The answer is a definite yes. After all, as in other fields of 

the biosciences, the constantly increasing flood of experi- 

mental data related to ageing research requires both ana- 

lysis and interpretation with the methods supplied by bio- 

informatics. This applies both to the biological process of 

ageing and research on diseases associated with ageing, 

since both phenomena are dauntingly complex. 

In the last few decades, new experimental ap- 

proaches have developed that can be grouped under 

the heading of automation, miniaturisation and par- 

allelisation. Huge amounts of biological data have 

been generated at unimaginable speed. The data have to 

be recorded, validated, analysed and interpreted. For this 

purpose, new methods are required whose development 

and application has led to the establishment of a new bio- 

logical discipline – bioinformatics. 

In 2001 research groups in Jena succeeded in gaining 

assistance from a funding initiative of the Federal Minis- 

try of Education and Research (BMBF) amounting to over 

eight million euros for the development of bioinformatics. 

This resulted in the founding of the Jena Centre for Bioin- 

formatics, the “JCB”. The new centre is an association of 

research groups from the Friedrich Schiller University, the 

Jena University of Applied Sciences, the Max Planck Insti- 

tute for Chemical Ecology, the Leibniz Institute for Natu- 

ral Product Research and Infection Biology (Hans Knöll In- 

stitute) and the Leibniz Institute for Age Research (Fritz 

Lipmann Institute). Also involved are the companies “Bio- 

Control”, “Clondiag Chip Technologies” and “Jenapharm”, 

all of which are located in Jena. 

The aim of the Jena Centre for Bioinformatics is to 

develop and link together the expertise available on 

the spot in the areas of bioinformatics and “computa- 

tional biology”. This has already improved and will 

continue to greatly improve both bioinformatics training 

and biomedical research and development. The focus of 

JCB research is “molecular communications processes in 

normal and pathological cell states”. Both this general ori- 

entation and numerous individual projects and methodo- 

logical developments will provide an excellent supple- 

ment to the focus of the research activities at the Fritz 

Lipmann Institute for Age Research, which is represented 

by an above-average number of groups at the Jena Centre 

for Bioinformatics.

Impulses for research 

In this way, the genome analysis lab of the Fritz Lip- 

mann Institute, directed by Matthias Platzer, has partici- 

pated in a number of national and international genome 

projects aimed at unravelling the human genome. Funda- 

mental work done by this group on so-called alternative 

splicing has been published. The junior group “Theoretical 

Systems Biology”, headed by Thomas Wilhelm at the Fritz 

Lipmann Institute, concentrated on the analysis of biologi- 

cal networks. Thomas has now moved to the Institute for 

Food Research at Norwich (U.K.) as systems biology group 

leader. The structural biology lab headed by Matthias 

Görlach has most recently been concerned with the clari- 

fication of the structure of virus proteins. The junior group 

“Theoretical Biophysics”, formerly led by Martin Zacharias, 

has contributed to the success of the Jena Centre for Bio- 

informatics. In the meantime Martin has been appointed 

professor at the International University of Bremen. Im- 

portant contributions have been made to improving the 

protein-docking procedure. In the biocomputing group 

under the leadership of Jürgen Sühnel, internationally rec- 

ognised databanks and analysis tools have been devel- 

oped. Amongst these are the Jena Library of Biological 

Macromolecules (www.fli-leibniz.de/IMAGE.html), the 

Jena Prokaryotic Genome Viewer (jpgv.fli-leibniz.de), the 

Spirochete Genome Browser (sgb.fli-leibniz.de) and the 

JCB Protein-Protein Interaction Website (www.fli-leibniz. 

de/jcb/). 

Active participation: Open house at the JCB 

Impulses for training 

In addition to carrying out research, the Jena Centre 

for Bioinformatics has also given a significant impulse to 

training. The core of bioinformatics training in Jena is the 

bioinformatics curriculum at the Friedrich Schiller Univer- 

sity. Additional training activities have included the intro- 

duction of bioinformatics components in the pharma-bio- 

technology and medical technology courses at the 

University of Applied Sciences and the JCB Centre for Post- 

graduate Training. 

Overall, the new centre has exerted a substantially 

positive effect on regional inter-linking and the interna- 

tional reputation of research in Jena. In 2005 and 2007, all 

national bioinformatics centres supported by the Federal 

Ministry BMBF were evaluated by a group of international 

experts in the field. The JCB made an excellent showing. 

In 2008 funding by the Federal Ministry will expire. There- 

fore, the JCB currently undergoes reorganisation to adapt 

to this new situation without losing the Centre’s positive 

effects. 

Author: Jürgen Sühnel 

Phone: 0049-3641-656200, 

E-mail: jsuehnel@fli-leibniz.de 

www.fli-leibniz.de/jcb/ 

25

26 Calkhoven Lab 

Translational Control of Gene Expression 

Regulated translation of specific mRNAs plays a pivotal role in the control of cell proliferation, 

differentiation and cellular functions. The laboratory of Cornelis Calkhoven is focussing on 

mechanisms of translation control, the signalling pathways involved and their physiological implica- 

tions by combining cell- and molecular-biological approaches and mouse models. 

Our work focuses on the regulation of expression of 

key transcription factors in cellular differentiation, prolif- 

eration and senescence at the mRNA-translation level. 

Recently, the aetiologies of several human diseases, in- 

cluding cancer, have been linked to mutations in genes of 

the translational control machinery or in cis-regulatory 

sequences of mRNAs. Accordingly, the development of 

novel therapeutic strategies targeting translational con- 

trol offers promising new prospects in the treatment of 

human diseases. 

uORF-mediated translation 

Small upstream open reading frames (uORFs) in C/EBP 

and SCL/TAL1 mRNAs serve as cis-regulatory elements 

controlling the site of translation initiation. By monitoring 

the activity of translation initiation factors (eIFs), they de- 

termine the ratio of expression of distinct protein iso- 

forms displaying different physiological functions. Hence, 

signal transduction pathways converging on the transla- 

tional machinery can affect cell fate through uORF-con- 

trolled translation. In order to examine the effect of aber- 

rant translation control of C/EBP mRNAs in vivo, 

C/EBPβ-uORF-deficient mice have been generated and 

C/EBPα-uORF-deficient mice are under construction. 

Deregulated translation of C/EBPα and 

-β protein isoform expression in cancer 

Translational deregulation of C/EBP isoform expres- 

sion through upregulation of translation initiation factor 

(eIF) activities results in disturbed adipocyte differentia- 

tion and cellular transformation in cell culture. In collabo- 

ration with Dr. Franziska Jundt and colleagues (Charité, 

Berlin) we have shown that inhibition of mTOR (mamma- 

lian target of rapamycin) mediated translational signalling 

by a pharmacological rapamycin derivate is strongly anti- 

proliferative in Hodgkin lymphoma (HL) and anaplastic 

large cell lymphoma (ALCL) cells and prevents lymph node 

metastasis of xenotransplanted lymphoma cells in mice. 

Rapamycin exerts its anti-tumour effect by the trans- 

lational down-regulation of pathologically high levels of 

the proliferation-promoting truncated C/EBPβ isoform. 

Accordingly, pharmacological inhibition of the mTOR 

pathway by rapamycin derivatives may represent a new 

treatment option in lymphoma therapy.

Translational protein isoforms of SCL/Tal1 

determine lineage outcome of primary 

bone-marrow cells 

The expression of different SCL/Tal1 protein isoforms is 

regulated by signal transduction pathways that modulate 

the function of the translation initiation factors (eIFs). A 

conserved small upstream open reading frame (uORF) in 

SCL transcripts acts as a cis–regulatory element control- 

ling isoform expression. At the onset of erythroid differ- 

entiation, truncated SCL protein isoforms arise by alterna- 

tive translation initiation and favour the erythroid 

lineage. In comparison, full-length SCL proteins are 

more efficient in enhancing megakaryocytic dif- 

ferentiation. Our studies have revealed transla- 

tional controlled gene expression as a novel mech- 

anism regulating haematopoietic lineage outcome. 

Metabolic signalling pathways 

control C/EBP isoform 

expression 

Nutrient and energy signalling through mTOR 

(mammalian target of rapamycin) and AMPK (AMP-acti- 

vated protein kinase) converge on the translational con- 

trol machinery to adapt global and specific mRNA transla- 

tion to the energy and nutritional status of the cell. 

Cancer cells have overcome the usual restriction by nutri- 

ents and energy checkpoints, enabling them to prolifer- 

ate and maintain high metabolic rates even when nutri- 

Insights into the inside of the cell, 

in the front: an adipocyte 

ents and energy are in short supply. In addition, the mTOR 

kinase pathway has been shown to regulate insulin sensi- 

tivity and is believed to be involved in the development of 

type II diabetes. Our recent studies show that C/EBP trans- 

lation is strongly affected by the availability of nutrients. 

Interestingly, C/EBPα and -β are key regulators of genes 

involved in energy, glucose and lipid metabolism. There- 

fore we are investigating how C/EBP translation is inte- 

grated into the growth factor and nutrient mTOR signal- 

ling network and whether the resulting adaptation of 

C/EBP activity leads to a response in metabolic gene 

regulation and cell growth. 

C/EBP regulation in senescence and 

ageing 

Recently, several studies have linked C/EBPα and -β 

function to senescence and ageing. Interestingly, ad- 

equate expression of IGF-1 and insulin receptors, 

which regulate longevity in a conserved manner 

throughout species, depends on C/EBP transcrip- 

tion-factor activity. In addition, C/EBPα interaction 

with the chromatin-remodelling factor Brahma is associ- 

ated with a compromised regeneration capacity in the 

elderly liver. Our scientific aims are to reveal the role of 

translationally controlled C/EBP expression in replicative 

senescence and ageing, to identify co-factors and co-regu- 

lators in C/EBP isoform function and to study the signal 

transduction pathways involved. C/EBPα-uORF and 

C/EBPβ-uORF-deficient mice will be used to study ageing 

and senescence in vivo. 

27

28 Calkhoven Lab 

A translational control reporter system 

(TCRS) 

Several proteins of the translational control signalling 

network and machinery, as well as translationally control- 

led genes, are implicated in oncogenic, neurological, in- 

flammatory and metabolic disorders. It is anticipated that 

translational control in vertebrate development and dis- 

ease will prove to be of greater importance than previ- 

ously thought and that its exploration may provide novel 

targets for therapy. Accordingly, we have created a trans- 

lational control reporter system (TCRS) designed to iden- 

tify such agents and aid the development of novel thera- 

peutic strategies in treating cancer and other human 

diseases. 

Histological staining of liver with Sudan III: staining of fat droplets 

(fatty liver) 

The distribution and 

amount of proteins in the 

cell can be shown using 

immunofluorescence. 

Lab members: Cornelis Calkhoven, Christine Müller, Laura Maria 

Zidek, Sabrina Schubert, Sandra Schreiber, Thomas Niemietz. 

Not pictured: Anna Bremer, Götz Hartleben 

Author: Cornelis Calkhoven 

Phone: 0049-3641–656005 

E-mail: calkhoven@fli-leibniz.de

Cellerino Lab 

Using a Short-Lived Fish to Investigate the Biological 

Mechanisms Controlling Lifespan 

Ageing research involves testing the effects of experimental or genetic manipulations on lifespan 

and age-related pathologies. However, the lifespan of the available model systems represent 

a hurdle that cannot be overcome. Alessandro Cellerino presents N. furzeri as a new model 

system for age research, its advantages over other models, and first data of his research projects. 

A suitable model for age research 

The annual fish Nothobranchius furzeri inhabits 

ephemeral pools in semi-desert areas with scarce and er- 

ratic precipitation. It adapts to the routine drying of the 

environment by evolving desiccation-resistant eggs that 

can survive for one or more years. Due to the very short 

duration of the rain season, the natural lifespan of these 

fish is only a few months and their captive lifespan is sim- 

ilarly brief. The inbred strain Gona Re Zhou develops from 

a larva to a sexually mature adult in 3-4 weeks, with me- 

dian survival of 8.5 weeks under laboratory conditions. 

There are three reasons why this model is particularly 

suitable for studies on ageing: 

First, it displays highly accelerated rates in a series of 

conserved age-related markers typical of vertebrates: i) 

Reduction of locomotion, as quantified by open-field ex- 

ploration, a standard behavioural test used for rodents 

that displays age-dependent decline. Such greatly acceler- 

ated decline is also present in N.furzeri and is already de- 

tectable at the age of 9 weeks. ii) Cognitive decline, as re- 

flected by an operant conditioning protocol (shuttlebox). 

Young fish (5 weeks old) show significantly higher per- 

formance than old fish (9 weeks old). This simple test re- 

veals an age-dependent learning deficit in N. furzeri. iii) 

Neurodegeneration, as demonstrated in the brain using 

the specific dye Fluoro-JadeB. iv) Histological markers in 

peripheral organs: Lipofuscin (LF), the autofluorescent 

„ageing pigment“, displays age-dependent accumulation 

in the liver, while senescence-associated beta-galactosi- 

dase (SA-β-Gal), an age-related marker in humans and 

zebrafish, is detectable at 9 weeks of age. 

Secondly, longevity and the expression of age-related 

markers are modulated by treatments known to increase 

lifespan in other model organisms. A reduction of water 

temperature by only 3°C induces an increase of median 

lifespan from 9 to 10 weeks and of maximum lifespan 

from 11 to 12.5 weeks. Simultaneously, it prevents the ex- 

pression of age-related locomotor and learning deficits at 

9 weeks and reduces the accumulation of the age-related 

markers. These data strongly indicate that short lifespan 

in N. furzeri is not pathologically induced but linked to ac- 

celeration of ageing, possibly as result of adaptation to its 

particular environment. 

29

30 Cellerino Lab 

A view of the FLI Nothobranchius facility, where the fish 

are grown to perform pharmacological studies. 

Thirdly, N. furzeri can be raised in relatively high num- 

bers and this has enabled us to perform finely graded, 

age-dependent survival studies. It is possible to analyse 

more than a hundred experimental fish, deriving not only 

median and maximum survival but also the parameters of 

mortality curves such as ageing rates and age-independ- 

ent mortality. This type of analysis is important in dis- 

cerning whether a treatment reduces mortality risk 

equally at all ages or retards the age-related acceleration 

of mortality so that the difference in mortality becomes 

progressively larger with increasing age. 

In summary, this model enables us to measure the ef- 

fects of experimental manipulations on mortality and 

age-related traits using a fraction of the time (and fund- 

ing) that would be required to perform the same experi- 

ments on zebrafish or mice. 

Effects of pharmacological interventions 

on lifespan and age-related pathologies 

We have already used N.furzeri to test the effects of 

life-long treatment with resveratrol on longevity and age- 

related markers. In N.furzeri, resveratrol induced a dose- 

dependent life extension of up to 60% increase in maxi- 

mum lifespan. This life-extension effect was linked to 

retardation in the onset of age-dependent cognitive and 

locomotive deficits and prevention of age-dependent 

neuronal degeneration. These results identify resveratrol 

as the first small molecule able to increase lifespan in ani- 

mals as diverse as C. elegans, Drosophila and a fish. In fu- 

A fully developed embryo of Nothobranchius furzeri in the 

dry substrate waiting to hatch. If water is dropped onto the 

egg at this stage, the embryo produces proteases to free itself 

from the chorion and in a few hours a newborn fry will swim. 

ture, we will be studying both small molecules targeting 

specific biochemical pathways and candidate drugs, 

measuring their effects on lifespan, age-related patholo- 

gies and cognitive decline. 

N.furzeri also represents a model system for investi- 

gating the genetic basis of lifespan. This project, de- 

scribed on page 21, is conducted in close-collaboration 

with the two laboratories of Matthias Platzer and 

Christoph Englert at FLI. 

Author: Alessandro Cellerino 

Phone: 0049-3641-656439 

E-mail: acellerino@fli-leibniz.de 

Lab members: Eva Terzibasi, Alessandro Cellerino

All important functions 

of a cell are coordinated 

in its „command centre“, 

the nucleus. 

Early Ageing and Premature Death: 

When Cellular Control Systems Fail 

During cell division (mitosis) the centromere/kineto- 

chore complex mediates the link between chromosomes 

and microtubuli of the spindle apparatus. Faithful guard- 

ing of this interaction is crucial for the precise distribution 

of the genetic material among the two daughter cells, a 

process called chromosome segregation. In higher organ- 

isms the correct distribution of the chromosomes is pre- 

cisely controlled by proteins of the mitotic „checkpoint“. 

Failure of this control unit causes aneuploidy and may re- 

sult in cancer. The aim of the lab is to describe the mecha- 

nisms essential for chromosome segregation and the 

functions of the proteins involved. 

Promyelocytic leukaemia nuclear bodies (PML NBs) 

locally accumulate a dynamic range of specific proteins, 

many of which are key regulators of cellular senescence, 

the DNA damage response, and transcriptional regulation. 

The precise function of PML NBs in these processes are 

not understood. The lab analyses the function of the PML 

protein and the dynamic composition of PML NBs in nor- 

mal and perturbed human cells. 

Diekmann Lab 

Important cell functions are organised and controlled in the cell nucleus. This organelle ensures 

correct chromosome distribution during cell division and proper repair of damaged sites in the 

DNA molecule. The major focus of research in Stephan Diekmann‘s laboratory are centromeres and 

promyelocytic leukaemia (PML) nuclear bodies 

The centromere/kinetochore complex: 

always in focus 

The number of proteins performing the complex task 

of chromosome segregation is surprisingly large. We are 

cloning all these proteins in order to make them visible in 

the cell interior. This enables us to determine their proper- 

ties in their natural environment and to draw conclusions 

about their molecular environment, reaction partners and 

motility. We establish where and when the proteins per- 

form certain functions and whether or not they deal with 

different tasks in different places and at different times. 

Complex processes of this kind can now be studied in 

living cells, the most natural setting for a protein. 

To maintain genomic stability, check-point mecha- 

nisms supervise whether or not the cell is in good meta- 

bolic shape, whether all conditions are fulfilled to start 

DNA replication, whether replication was successful and 

complete, whether each of the chromosomes has been at- 

tached to the mitotic spindle, and whether distribution of 

31

32 Diekmann Lab 

One of the most important events in the life cycle 

of a single cell is cell division, a complex 

mechanism following a precise choreography. The 

correct distribution of DNA to the daughter cells is 

of particular importance for their future fate. 

the chromosomes to the newly arising daughter cells was 

successful. In case of an accident, cells halt the cell cycle 

and try to repair or remove the defect. One major regula- 

tor of cell division is the anaphase promoting complex/cy- 

closome (APC/C), dysfunction of which causes improper 

sister chromatid separation. The APC/C complex consists 

of at least 12 known core subunits and its activity is to de- 

lay anaphase onset until all chromosomes are correctly at- 

tached to the mitotic spindle. When completed, APC/C 

chemically modifies the cohesin inhibitor securin, leading 

to cohesin degradation followed by chromatid separation. 

Unattached kinetochores inhibit, through a complex in- 

cluding the protein BubR1, the E3 ubiquitin ligase activity 

of APC/C. We clone all proteins involved and study their 

interaction and interplay by life cell imaging. 

As an example, we study the checkpoint protein 

BubR1. If this protein is not available in sufficient quanti- 

ties, DNA will not be correctly distributed between the 

two daughter cells during mitosis. BubR1 depletion to 

5-10% of normal levels results in an ageing phenotype in 

mice, and, in cells, causes mitotic checkpoint failure as 

well as a compromised response to DNA damage, and 

early cellular senescence. Humans with BubR1 mutations 

show phenotypes of mosaic-variegated aneuploidy. To 

test the hypothesis that checkpoint protein function requires 

expression and availability above a threshold value 

in order to ensure the function of the mitotic checkpoint 

complex, BubR1 will be studied by live cell fluorescence 

microscopy. The hope is to elucidate function and interplay 

of mitotic checkpoint proteins, and maybe we can 

identify processes how they influence human ageing. Our 

objective is to understand the molecular processes underlying 

BubR1 deficiency-induced premature ageing. 

Mysterious nuclar bodies 

The cell nucleus accommodates not only chromosomes 

but also a number of so-called nuclear bodies (NBs). The 

nucleolus represents the best known of the NBs and we 

have detailed textbook knowledge on how the nucleolus 

functions in ribosome biogenesis. On the other hand, the 

precise function(s) of PML bodies is still enigmatic. 

The abbreviation PML stands for “promyelocytic 

leukaemia”, a form of blood cancer. PML is also the term 

used for the protein that figures most prominently in this 

macromolecular assembly, which contains 9 permanent 

and more than 60 transient protein members apart from 

PML. Strikingly, in cells of patients suffering from promyelocytic 

leukaemia the PML bodies are disrupted and a certain 

type of blood cells can no longer form. 

The current model suggests that PML NBs serve as sites 

for specific nuclear protein modification, sequesteration 

and/or complex assembly. PML NBs specifically bind to 

DNA double-strand breaks, suggesting that they are capable 

of sensing these dangerous lesions. During the cellular 

senescence program PML bodies specifically accumulate 

proteins which mediate this process. In addition, these 

structures are spatially associated with specific genomic regions, 

which contain genes that might be under the transcriptional 

control of PML. Our aim is to establish the structure 

and function of PML nuclear bodies in these processes.

To this end we mark the protein components of these 

structures to make them visible under the microscope. 

This enables us to identify the structure and composition 

of these assemblies more accurately. In addition, we com- 

bine microscopy with biophysical methods to find out 

how quickly these proteins move in a living cell, how long 

the proteins stay at the nuclear body, what other proteins 

they react with and how they do their biochemical work 

on the DNA. 

Different fluorescence techniques allow to 

visualise different nuclear bodies, which are 

sub-nuclear compartments within the cell 

nucleus. 

In-house collaborations: 

The checkpoint mechanisms of the centromere/kineto- 

chor complex ascertain that DNA replication (Große lab) 

and DNA repair (Wang lab) have been properly completed. 

The protein which is defective in the human Nijmegen 

Breakage syndrome (NBS1; Wang lab) has been found to 

be associated with the PML bodies. The putative func- 

tional links are studied in collaboration. 

In a broader sense, the functional analyses of the cen- 

tromere and PML nuclear bodies shall help us to under- 

stand the basic principles underlying the functioning of 

the cell nucleus in normal and defective cells. 

Lab members: Stephan Diekmann, Christian Hoischen, Marianne 

Koch, Sabine Ohndorf, Sandra Münch, Stefanie Weidtkamp-Peters, 

Sylke Pfeifer, Britta Reichenbächer, Adelheid Diete, Tobias Ulbricht, 

Christiane Hirsch, Peter Hemmerich. Not pictured: Daniela Hellwig, 

Christian Weber 

Authors: Stephan Diekmann, Peter Hemmerich, Christian Hoischen 

Phone: 0049-3641-656260 

E-mail: diekmann@fli-leibniz.de 

33

34 Englert Lab 

From Genes to Organs: 

How Genes Control Development 

Very little is known about the way in which organs form, which genes control this complex process, 

how they do so and the way in which these genes are regulated. With reference to the 

development of kidneys and gonads, Christoph Englert is investigating how genes and their gene 

products contribute to “organogenesis” and the malformations and illnesses that ensue if the genes 

are unable to perform their organogenetic functions. 

Apart from infectious diseases, most of the illnesses to 

which the human body is subject are caused by mutations, 

i.e. changes to hereditary material. How these mutations 

ultimately affect the structure or function of an organ is 

still largely unknown. To achieve a better understanding 

of these mechanisms we first need to discover the princi- 

ples underlying organ formation. With reference to devel- 

opment in the urogenital area of the body (the formation 

of the kidneys and the gonads), we are investigating the 

genes and gene products (proteins) that control the proc- 

ess of “organogenesis”. Mutations of these genes are re- 

sponsible for numerous human diseases. 

One of the genes involved in organogenesis is the 

“Wt1” gene and its gene product, the “Wilms tumour pro- 

tein Wt1”. This protein is indispensable for normal devel- 

opment of the kidneys and gonads. If the Wt1 protein is 

missing or not functioning properly, the result is the 

“Wilms tumour”, a form of kidney cancer. One child in 

every 10,000 is affected by this tumour, other malforma- 

tions in the urogenital area have the same causes. We do 

not yet know how the Wt1 protein causes the organs to 

develop. It performs at least part of its functions as a 

“transcription factor”, which means that it switches genes 

on or off. 

Of mice and fish 

At present we are using mice and zebrafish to investi- 

gate precisely which genes are switched on or off by Wt1. 

For this purpose we compare normal tissue with tissues in 

which the Wt1 protein has been deactivated. In this way 

we have already identified a number of Wt1’s target 

genes. We know that one of these genes is responsible for 

the production of an important receptor. This receptor is 

part of the signalling pathway phasing out “female” struc- 

tures during the development of male embryos. This ob- 

servation explains the specific kind of hermaphroditism 

sometimes leading to a complete sex reversal that is fre- 

quently encountered in patients with Wt1 mutations.

A zebrafish embryo at 17 hours post fertilization. The eyes 

(bottom left) as well as the somites (right) are already 

distinguishable. From the latter bones, muscle and skin 

develop. 

In future we intend to study the significance Wt1 has 

for the development of other organs. For this purpose we 

have produced so-called knockout mice in which the Wt1 

gene has been altered so that it can be switched off in 

certain tissues and at certain times. In this way we can 

recognise the function of the gene or its protein in de- 

fined organs. 

We also use zebrafish to study the function and regu- 

lation of Wt1. As model organisms these fish have a 

number of advantages. They are relatively easy to keep 

and to manipulate genetically, they produce a large 

number of offspring and their embryos are transparent. 

In our initial experiments we established that, unlike 

many other creatures studied previously, zebrafish have 

two Wt1 genes. Though their activities overlap, these 

genes are clearly distinguished from one another. 

To find out how the two Wt1 genes are 

regulated, we bred fish in which the con- 

trol region of the relevant Wt1 gene 

was fused with a gene for green 

fluorescent protein (GFP). Accord- 

ingly, the fish embryos light up 

green where the corresponding 

Wt1 gene is active. With the help of 

fish like these we intend in future to 

analyse the factors responsible for 

the temporally and spatially specific 

activity of the two Wt1 genes. 

The activity of a gene that is important for kidney and gonad 

development (Anti Müllerian hormone receptor 2, Amhr2) is 

controlled by a protein called “Wt1”. In a normal 11-day old mouse 

embryo (left) Amhr2 is active in the developing gonads (dark blue 

color). When Wt1 is inactivated, Amhr2 is not turned on (right) 

and gonad development stagnates. 

wt1a α-tropomyosin 

wt1b α-tropomyosin 

Finally, we also intend to use these fish to study the 

function that Wt1 proteins perform. Initial experiments 

have indicated that the two Wt1 proteins are essential for 

the normal development of the kidneys. If one of the two 

genes is deactivated, the kidneys either cannot develop 

at all or malformed cystic kidneys are the outcome. 

A number of the physically recognisable changes we have 

observed in these fish are very similar to disorders found 

in humans. This underlines the significance of zebrafish 

as a model for the study of human diseases. 

wt1a 

wt1b 

15 hpf 15 hpf 15 hpf 

A 12-day old mouse embryo: The dark blue 

colour indicates the activity of a particular 

gene that encodes a transcription factor. The 

latter is a protein that can switch on and off 

other genes. This particular gene is active in the 

limbs as well as in the head. 

35

36 Englert Lab 

A 35-hour old zebrafish is oriented such that the head is on the 

right and the tail on the left. At this age the body is still 

transparent and the inner organs can be seen; in this case the 

kidneys are visualised by fluorescence microscopy. 

“Eya” – an old gene family 

Other genes called Pax, Six and Eya are responsible for 

the development of the kidneys and other tissues and 

organs like muscles, the eyes and hearing. Mutations of 

the human Eya1 gene lead to pathogenic changes affect- 

ing hearing, the kidneys and other parts of the body. In 

evolutionary terms the genes of the Eya family have been 

well conserved. They are very old and function in many 

different organisms, including plants, fruit flies and mam- 

mals. They regulate cell differentiation (the maturation of 

embryonic cells to form a specific cell type with a specific 

function), proliferation and survival. In biochemical terms, 

the Eya proteins unite the activities of factors influencing 

transcription (the reading-off of genes from the DNA) 

with the activities of signalling molecules and enzymes 

(phosphatases). 

To achieve a better understanding of the various func- 

tions of Eya1 we are attempting to identify interaction 

partners and target proteins of the phosphatases. These 

experiments are being performed initially on cell cultures. 

Subsequently we intend to investigate how the enzymatic 

activity of Eya1 is connected to its biological activity and 

thus identify which of the many biochemical functions 

are indispensable for organ formation. 

“Eya1”, a member of the Eya family of proteins is translocated 

into the nucleus by Six proteins. In the absence of Six, Eya1 is 

distributed throughout the cell (left), while in the presence of Six, 

Eya1 is exclusively in the nucleus. In order to visualise Eya1, it has 

been tagged with a fluorescent label. 

Molecular basis of ageing 

In a common effort with the labs of Matthias Platzer 

and Alessandro Cellerino we have established fish colo- 

nies of the species Nothobranchius at FLI. The goal is to 

establish this short-lived fish as a novel model for ageing 

research. Our lab is particularly interested in identifying 

molecular pathways that control ageing. To that end we 

are analysing ageing-associated changes in gene expres- 

sion and are studying the role of reactive oxygen species 

(ROS) as well as of chromosomal end structures, the so- 

called telomeres in the ageing process. 

Author: Christoph Englert 

Phone: 0049-3641-656042 

E-mail: cenglert@fli-leibniz.de 

Lab members: Kathrin Landgraf, Michael Graf, Amna Musharraf, 

Birgit Besenbeck, Claudia Reichardt, Christina Ebert, Christoph Englert, 

Dagmar Kruspe, Eric Rivera-Milla, Nils Hartmann, Birgit Perner, 

Peter Reinhardt, Ronald Schmidt, Andreas Boland, Ralph Sierig, 

Martin Franke, Frank Bollig. Not pictured: Bianca Lanick

Structure and Formation of Amyloid Fibrils 

Biophysical principles of amyloid formation 

and aggregation 

We have been able to show that the formation of amy- 

loid fibrils is not a property specific to those polypeptide 

sequences that form such structures inside the human 

body. In fact, these fibrils can also form in vitro from 

polypeptide chain sequences not known to do so in vivo. 

Examples of such behaviour are myoglobin and polyamino 

acids. These and other data suggest that amyloid fibrils 

represent a generic structural form of the polypeptide 

chain that is primarily determined by the invariant inter- 

actions of the polypeptide main-chain. However, side 

Fändrich Lab 

Amyloid fibrils are fibrillar polypeptide aggregates that possess a cross-β structure. These fibrils 

can occur inside the human body and are associated with ageing and disease. The best-known 

disorders involving amyloid formation are Alzheimer‘s and Creutzfeldt-Jakob disease, where deposits 

of such fibrils occur inside the brain. In addition, amyloid fibrils can occur also outside the brain and 

in most, if not all tissues. Here they are formed from different polypeptide sequences, including the 

serum amyloid A protein or medin. The ability of natural polypeptide sequences to form amyloid 

fibrils is particularly remarkable, given that the structure of the amyloid fibrils may be radically dif- 

ferent from the native structures of the same polypeptide chain. The main aim of the lab headed by 

Marcus Fändrich is to contribute to a better understanding of the structure of amyloid fibrils and 

the principles underlying their formation. 

Deposits in the brain with serious consequences 

chains and the physico-chemical environment have pro- 

found effects on the kinetic partitioning between amyloid 

fibrils and other structural forms of the polypeptide chain, 

as well as on the thermodynamics of aggregation. Using 

the Alzheimer’s Aβ(1-40) peptide as a model system, we 

have systematically explored the thermodynamic and ki- 

netic consequences of mutation and different side-chain 

properties on the aggregation process. We found that the 

speed at which different polypeptide chains nucleate and 

elongate depends very much on the extent to which the 

formation of these aggregates is thermodynamically fa- 

vourable. 

37

38 Fändrich Lab 

Thin-layer chromatography of lipid extracts 

from amyloidotic tissue (AA, AL, ATTR) and 

normal tissue. Various lipids are analysed, 

CH: cholesterol, SM: sphingomyelin 

Structure of amyloid fibrils from 

Alzheimer‘s Aβ peptide 

All amyloid fibrils are defined by the presence of a com- 

mon structural motif, termed the cross-β conformation. 

Accordingly, knowledge of fibril structure is a prerequisite 

for the understanding of the forces and biophysical princi- 

ples stabilising these states and for structure-based meth- 

ods designed to interfere with their formation. With 

Nikolaus Grigorieff from Brandeis University we are using 

circular dichroism and infrared spectroscopy, as well as 

high-resolution cryo-electron microscopy to study the 

structure of amyloid fibrils. Cryo-electron microscopy has 

been particularly useful in enabling us to reconstruct the 

Alzheimer’s Aβ(1-40) amyloid fibril and to analyse the way 

in which the peptide molecules are packed into the fibril 

quaternary structure. We found that the fibril is a left- 

handed helix formed from two protofilaments. Recently, 

we were able to refine the resolution of this structure to 

Amyloid fibrils of the brain, visualised by 

electron microscopy (left). 

Reconstruction of a single amyloid fibril, 

based on cryo-electron microscopy (right). 

less than 10 Å, one of the highest resolutions achieved so 

far for amyloid fibrils. It is now clear that the fibril mor- 

phology examined in this way is constructed from proto- 

filaments comprising a central structural spine of two 

closely packed cross β-sheets. The pairing of these β- 

sheets in the protofilament core resembles the one in re- 

cently proposed steric zipper structures. 

Conformational antibody domains for 

studying amyloid formation 

From the recombinant library of camelid VHH-anti- 

body domains established by the group of Dr. Uwe Horn 

(Hans-Knöll-Institute) we were able to select, by phage 

display, an antibody domain termed B10 that acts in a 

conformationally sensitive manner: it interacts strongly 

with the mature amyloid fibrils from Alzheimer’s Aβ pep- 

tide but not with disaggregated forms of this peptide or 

specific Aβ oligomers. Hence, B10 differs from sequence- 

specific antibodies such as 22C4, which recognise both the 

monomeric Aβ peptide and Aβ fibrils. Using B10, we have 

examined twelve hippocampal sections from confirmed 

Alzheimer cases and ten age-matched controls. While 

Alzheimer cases show plaques when stained with B10, no 

such plaques were observed in the control samples. We 

conclude that the fibrils formed in vitro carry surface 

epitopes that are very similar to, if not identical with, 

amyloid fibrils from Alzheimer plaques. In vitro aggrega- 

tion assays of Aβ with or without B10 show that the anti- 

body domain potently interferes with the transition be- 

tween Aβ protofibrils and mature fibrils. B10 or B10 

derivatives may have future applications in amyloid de- 

tection and in therapeutic interference with the mecha- 

nism of amyloid formation.

Lipid interactions in the cellular mechanism 

of amyloid fibril formation 

Our analysis of amyloid fibrils derived from various 

human diseases (AA, ATTR, Aß2M, ALlambda and ALkappa 

amyloidosis) revealed that these are associated with a 

common lipid component that has a conserved chemical 

composition and is especially rich in cholesterol and 

sphingolipids, major components of cellular lipid rafts. 

This pattern is not notably affected by the purification 

procedure and no close lipid interactions can be detected 

when preformed fibrils are mixed with lipids. These data 

suggest the existence of common cellular mechanisms in 

the generation of different types of clinical amyloid de- 

posits. To study the possible relevance of amyloid-lipid in- 

teractions we have established several cellular amyloido- 

sis models, including one for AA amyloidosis. These 

cellular models lead to the formation of amyloid plaques 

that closely resemble the ones detected in the diseased 

Correlation between the thermodynamics 

(expressed with ΔG) and the kinetics of 

aggregation (expressed in the lag time t1 or 

elongation rate k) for valine 18 mutants of Aβ 

(1-40) and disease-related variants. 

tissue. Significantly, they contain the same lipid compo- 

nents that are characteristic of the disease-associated de- 

posits. We are currently using cell culture and in vitro as- 

says to study the potential biological relevance of the lipid 

component for amyloid fibril formation. These investiga- 

tions are partly conducted in conjunction with Christoph 

Kaether’s lab. 

Author: 

Marcus Fändrich 

Phone: 0049-3641-656306, 

E-mail: fandrich@fli-leibniz.de 

New adress: 

Max Planck Research Unit for Enzymology of Protein Folding 

Halle; Germany 

Phone 0049-345-5524970, 

E-mail: fandrich@enzyme-halle.mpg.de 

Amyloid plaque formation in the 

cell culture model of AA 

amyloidosis monitored by Congo 

red, left: bright field; right: dark 

field. 

Lab members: Jessica Meinhardt, Karin Wieligmann, Marcus Fändrich, 

Nicole Hartenstein, Michael Schuch, Christian Haupt, Carsten Sachse, 

Katharina Tepper, Ralf Friedrich, Karoline Klement 

39

40 Görlach Lab 

Biomolecular Matchmaking: 

How Molecules Contact Each Other 

Biological processes like growth, cell division or the repair of molecular damage require that 

the biomolecules involved establish specific contacts with each other. Severe diseases may be 

the outcome if contact of this kind is compromised or disrupted. Matthias Görlach explains how a 

modern biophysical technique called NMR spectroscopy can be used to investigate the three-dimen- 

sional structure of biomolecules and their mutual interaction. 

For biological processes to take their normal course 

there have to be mechanisms enabling biomolecules to 

specifically recognise each other. Disruptions of these in- 

teractions can lead to blockades or dysregulation operative 

in the onset of acute or chronic illnesses. The kind of inter- 

actions involved depends on the “fit” between molecular 

contact surfaces. In an attempt to understand the three- 

dimensional structure of the biomolecules involved we in- 

vestigate the molecules and their interaction with the help 

of nuclear magnetic resonance spectroscopy (NMR). In 

concrete terms, we concentrate on proteins and ribonu- 

cleic acids involved in anti-oxidative processes, degenera- 

tive neuromuscular disorders or playing a central role in 

cancer associated with virus infections. 

One of our projects centres around the repair of so- 

called oxidative damage caused by reactive oxygen species 

NMR structure of a viral RNA 

and modelled contact regions 

(red) between the RNA and a 

viral protein. 

(ROS). Such ROS molecules are formed either by normal 

metabolic processes or as a result of external factors. One 

way ROS cause damage is by chemically modifying amino 

acids, the building blocks of proteins. When damage of 

this kind accumulates the outcome is referred to as “mo- 

lecular ageing”. For example, the amino acid methionine 

is oxidised by ROS into methionine sulfoxide (MetSO). The 

repair of this damage is undertaken by special enzymes 

called MetSO reductases, MSR for short. These enzymes 

are crucial for the anti-oxidative response in cells of all or- 

ganisms. We know that fruit flies producing a surplus of 

such enzymes live considerably longer. 

MSR enzymes come in two classes (MSRA and MSRB). 

Their three-dimensional structure differs significantly. In 

addition, MSRBs in the human organism display apprecia- 

ble differences in their amino acid sequence (the order of

amino acids) outside the catalytically active centre. This 

suggests that certain areas of the MSRBs are responsible 

for certain interactions with substrate proteins, which 

makes it conceivable that MSRBs play a regulatory role 

under oxidative stress conditions. Accordingly, the objec- 

tive of our project is to understand the structure of hu- 

man MSRBs and their interactions with substrate proteins. 

Virus-associated cancers 

In this project we study two viruses, the human papil- 

loma virus (HPV) family and the hepatitis B virus. Papillo- 

maviruses cause both harmless warts and malignant con- 

ditions such as cervical tumours. This work is carried out 

in collaboration with the laboratories of Aspasia Ploubi- 

dou and Helen Morrison, who study the role of the cy- 

toskeleton in oncogenic progression and signalling path- 

ways in tumour cells, respectively. For a detailed report 

see page 18. 

Solid state NMR spectra of the ribonucleic acid (CUG)n. Background: Semithin section of a muscle fiber. 

Chronic infection with hepatitis B viruses (HBV) leads 

to cirrhosis of the liver or liver cell cancer. The virus in- 

vades the nucleus of the human cell with its DNA. The 

subsequent transcription of the virus DNA in the nucleus 

of the cell gives rise to intron-free messenger ribonucleic 

acids (mRNA) that have to be exported to the cytoplasm 

for the synthesis of viral proteins. All mRNAs of the virus 

contain a common component called the “HBV post-tran- 

scriptional regulatory element” (HPRE). Without it, mRNA 

transport from the cell nucleus to the cytoplasm is impos- 

sible. HPRE contains two characteristic signal structures 

(HSLα and HSLβ), which interact with cellular proteins 

during export. We have already succeeded in elucidating 

the structure of HSLα. At present we are investigating 

how it interacts with candidate cellular proteins. 

In its helical stem region, the stem-loop structure of 

HSLα contains a single bulged nucleotide (guanine resi- 

due). This residue is located close to a loop (the apical 

pentanucleotide loop or “penta-loop”) that adopts a novel 

structure. Such a combination of structural elements 

(loop with single adjacent bulged nucleotide) also serves 

in other systems as a specific recognition site for proteins. 

Structural features operative in a severe 

muscular disorder 

Inaccuracy in the duplication of the hereditary sub- 

stance, the DNA, prior to cell division can lead to the ex- 

pansion of short repetitive sequences in the DNA. Such 

expanded repeats are frequently associated with illnesses. 

For example, an expansion of CTG triplets in the gene for 

DM protein kinase (DMPK) leads to the emergence of long 

(CUG)n stretches in the 3’-noncoding region of DMPK 

mRNA. They bind “muscleblind” proteins (MBNL) and 

withdraw them from the active splice-factor pool. This in 

its turn leads to defects in the processing of pre-mRNAs. 

The result is muscle cell proteins that are unable to fulfil 

their function, which ultimately leads to a complex syn- 

drome known as “myotonic dystrophy” (DM1). DM1 is as- 

sociated, amongst other things, with myotonia (muscle 

“stiffness”), myasthenia (muscle weakness) and cataract 

41

42 Görlach Lab 

UV-light 

smoke 

Reactive oxygen species (ROS) cause damage to 

cellular structures and functions. Specific enzymes, 

the methionine sulfoxide reductases, are capable to 

repair certain damages. 

DNA-damage 

formation. Investigation of the structural basis of the in- 

teraction between MBNL and (CUG)n stretches via NMR 

in solution or X-ray structural analysis is impeded by the 

aggregation propensity of MBNL-(CUG)n complexes. 

Here, “magic angle spinning” solid-state NMR spectros- 

copy (MAS-NMR), which has recently developed into a 

powerful technique for elucidating biomolecular struc- 

tures, may provide a way out of the dilemma. At present 

MAS-NMR methods are available for proteins, but not for 

RNA. Accordingly, we are working on MAS-NMR tech- 

niques for the determination of the structure of RNA. In 

this way we have already succeeded in demonstrating the 

double-stranded helical structure of (CUG)n sections. 

Signal structures in viruses 

So-called enteroviruses cause various acute and 

chronic diseases. For example, the coxsackie virus B3 is re- 

sponsible for acute and chronic forms of myocarditis. The 

hereditary material of the viruses consists of ribonucleic 

acid (RNA), serving as messenger RNA and forming an ex- 

tensively structured untranslated 5’-region containing the 

internal ribosome entry site (IRES) and the 5’-terminal clo- 

verleaf (5’-CL). The latter functions as a signal for cellular 

and viral proteins in the assembly of the viral RNA replica- 

tion complex. An essential RNA-protein reaction in the 5’- 

CL takes place between the 3C domain of the viral 

polymerase precursor 3CDpro and a stem-loop structure 

(SLD) of the 5’-CL. Here it is mainly the apical loop of the 

SLD that is responsible for the specific interaction with 

3CDpro. This tetra-loop structure is remarkably conserved 

ROS 

repair 

protein- oxidation 

MSR 

protection 

in enteroviruses even though its sequence variability is 

surprisingly high. This analysis indicated that the specific 

recognition of the SLD signal structure by the 3C domain 

is governed by shape rather than by sequence specificity. 

This result has enabled us also to postulate a conceivable 

path for the transition between structurally unrelated 

tetra-loop families in the course of evolution. In toto, 

tetra-loops represent a fundamental structural element of 

RNA. 

Author: Matthias Görlach 

Phone: 0049-3641-656220 

E-mail: mago@fli-leibniz.de 

Lab members: Yvonne Ihle, Sabine Häfner, Oliver Ohlenschläger, 

Michela Carella, Christiane Hirsch, Hansjörg Leppert, Angelika Heller, 

Thomas Seiboth, Ramadurai Ramachandran, Marina Baum, Christian 

Herbst, Christine Kamperdick, Matthias Görlach, Georg Peiter, Kerstin 

Riedel, Matthias Nestler. Not pictured: Anika Kirschstein

The best known process for the fusion of genetic properties 

of two cell types is sexual reproduction. Other cell types 

which do not fuse spontaneously, can be induced to fuse 

under microscopic control by a laser microbeam. Upper row: 

Fusion of protoplasts for plant breeding. Lower row: Fusion 

of immune cells with the potential to produce antibodies. 

Getting Sorted: Functional Molecule Blocks 

Greulich Lab 

At first glance, the countless molecules acting and interacting in the interior of the cell appear 

to be an impenetrable tangle. Karl Otto Greulich explains how scientists make sense of this 

apparent chaos and outlines the surprising insights into the life of the cell that can be gained by 

grouping molecular processes into larger, “functional blocks”. Techniques of this kind may one day 

enable us to understand the changes undergone by meaningful molecular units during the ageing 

process. 

One present objective of research in cell biology is to 

understand what goes on in the living cell at the molecu- 

lar level. We can see how ambitious this goal is if we bear 

the following figures in mind: the human genome con- 

tains the blueprint for over 20,000 different molecules; 

an average cell uses about 10,000 of these molecules, but 

a liver cell draws upon an entirely different set of genes 

from, for example, a cell in the pancreas. 

At present, scientists all over the world are investigat- 

ing the various ways in which different molecules interact 

in the interior of the cell. To do so, they attempt to group 

these interactions into “functional blocks”. This perspec- 

tive drastically reduces the number of possible interac- 

tions. To see why, we can draw an analogy with the way 

in which pairings are selected in a soccer tournament. If 

all the 16 soccer teams taking part in the European Cham- 

pionships were paired off freely, the number of possible 

encounters for the first preliminary round alone would be 

43,680. But if each group is headed by a seeded team and 

the pairings are taken from three to six “pots” containing 

previously selected teams, then the number of potential 

pairings declines dramatically. 

A similar approach can be used to impose order on the 

molecules in the interior of the cell. Here too, the tangle 

of molecular interactions and potential molecular reac- 

tions become much easier to survey if we arrange them 

into sets that we call “functional blocks”. Such blocks 

may be metabolic pathways, such as glycolysis (the 

breakdown of sugar), or reaction pathways like DNA re- 

pair by enzymes. Another functional block is the one 

grouping interactions typical for the tumour suppressor 

molecule p53. This growth-regulating protein is able to 

curb the further proliferation of cancer cells and has al- 

most 100 reaction partners. 

To identify interesting connections with a compara- 

tively low degree of effort, it is best to desist from doing 

the measuring work oneself and to trust to the skill of 

colleagues all over the world in performing (on average) 

correct measurements despite all the experimental pit- 

43

44 Greulich Lab 

Damaged DNA inside the cell nucleus can be visualized with the COMET assay. The cell is embedded in an electrophoresis gel 

and an electric field is applied. The negatively charged DNA migrates towards the positive pole of the field. Highly fractionated 

DNA migrates further than less fragmented DNA. Left: Nucleus with moderate DNA damage. Middle: DNA damage is visible. 

Right: After long time of electrophoresis and change of field direction, even undamaged chromosomes are stretched and can 

be observed directly. Such a direct view on stretched chromosomes is hardly available with other techniques. 

falls and potential errors that can interfere with their 

work. Access to very many measurement data published 

world-wide is almost always possible if we restrict our in- 

quiries to the amount of a given molecule present in a 

given tissue (or, in scientific parlance, how highly ex- 

pressed it is). An excellent source for information of this 

kind is the “dbEST” database published by the National 

Institute of Health (NHI) in the United States. It contains 

data on 51 different healthy types of tissue and the cor- 

responding cancer tissues. 

The figure on page 45 illustrates a sur- 

prisingly simple outcome discovered in 

this way. The two columns in the figure 

indicate how high the sum of expression 

levels of a selection of 63 molecules inter- 

acting with the tumour suppressor mole- 

cule p53 is. The left-hand column shows 

the expression pattern in liver tissue, 

while the column on the right shows the 

expression pattern in pancreatic tissue. It 

is discernible at a glance that a gene 

called “STAT1” (violet) has a considerable 

share in the overall height of the column 

for liver tissue. By contrast, it is difficult 

to identify it in pancreatic tissue. Overall 

there appear to be far more genes in the 

liver than in the pancreas. The dominant 

gene in the pancreas is “Ddr1” (orange). 

Taken on their own, these observations 

are not particularly surprising. After all, 

the expression patterns for cells perform- 

ing different tasks are themselves bound 

Direct view into DNA repair: Using a laser 

microbeam, DNA double strand breaks are 

induced, here in the form F(ritz) L(ipmann) 

I(nstitute). The DNA repair, which starts 

after a few seconds, is visualized with a 

GFP labelled protein, which is recruited to 

the site of DNA damage and then activates 

a whole functional molecule block. 

to differ. The really surprising thing is the height of the 

columns. Though they are made up of different genes, 

they are almost equally high. This remains true if we com- 

pare not only the liver and the pancreas but 24 different 

types of tissue. Though expression for single genes may 

deviate by a factor of 100, the columns for all 24 types of 

tissue are similar in height, with a standard deviation of 

33 percent. If we then include the corresponding cancer 

tissues in our observations, we find columns heightened 

by a factor of 1.4 with a standard deviation of 16 percent. 

In other words, cancer both increases the cumulative ex- 

pression values (the height of the col- 

umns) and unifies them. Using these 

principles, a classifier has been devel- 

oped in cooperation with the Sühnel lab, 

which safely allows to distinguish colon 

cancer tissue from normal tissue on the 

basis of the 62 gene products which in- 

teract with the p53 molecule. Presently, 

it is shown, together with the Diekmann 

lab, that the genes for the different vari- 

ants of the centromere proteins CENP 

are expressed in a very tissue specific 

manner. 

We have established this fact not 

only in connection with the molecules 

interacting with p53 but also for glycoly- 

sis and DNA repair enzymes. In the case 

of the repair enzymes, we first identified 

similarities or differences in the expres- 

sion of single enzymes or whole repair 

chains in different kinds of tissue. At 

present we are testing this finding ex-

Interactions of the p53 tumour suppressor: Both columns show for each individual molecule 

(slice of the column) the expression level of a choice of 62 molecules which interact with p53. 

The left column gives the expression pattern in liver, the right one in pancreas. In liver, much more 

different genes are expressed than in pancreas, where the gene Ddr1 highly contributes to the height of 

the column. In spite of the very different composition, both columns have almost the same height. 

perimentally with fluorescent, so-called GFP constructs of 

the repair enzymes. These experiments are performed in 

tight cooperation with the Wang lab. To gain a dynamic 

view of DNA repair we use laser microscopy techniques. 

These enable us to systematically cause DNA damage in 

the cell nucleus and subsequently to study repair mecha- 

nisms microscopically with temporal resolution in the 

vicinity of one second. In these areas we closely interact 

with the Diekmann and Wang laboratories. 

Our investigations are designed to show the extent to 

which the understanding of molecular connections in the 

cell can be simplified by observing gene blocks and their 

interactions. One day this will also enable us to under- 

stand the changes undergone by functional blocks in the 

ageing process. 

Author: Karl Otto Greulich 

Phone: 0049-3641-656400 

E-mail: kog@fli-leibniz.de 

Lab members: Paulius Grigaravicius, Sabine Hoffmann, Kerstin 

Dreblow, Norman Gerstner, Anandhakumar Jayamani, Nikolina 

Kalchishkova, Gabriele Günther, Shamci Monajembashi, Roland 

Stracke, Kornelia Haus, Karl Otto Greulich, Marina Wollmann, 

Leo Wollweber, Silke Schulz, Konrad Böhm, Maria Yosifova Radeva. 

Not pictured: Teresa Keining, Christine Beck 

45

46 Große Lab 

An Elegant Balancing Act: 

How Cells Maintain their Genetic Stability 

Inner stability is as important for cells as for anything else. If cells lose their genetic balance, 

severe disorders like cancer may be the outcome. A loss of genetic stability can also be observed 

when cells age. Frank Große outlines the sophisticated repair mechanisms with which cells maintain 

their genetic stability even in the face of recurrent damage. He also describes what happens when 

cellular repair proteins fall down on the job. 

In the life of every organism there are recurrent cases 

of DNA damage that interferes with effective replication 

and transcription. Damage of this kind, which can occur 

during the DNA duplication process and/or the transcrip- 

tion of DNA information into RNA, has to be recognised 

and reported to the cell’s repair apparatus. Repair failure 

due to the absence of an important enzyme leads to an 

accretion of mutations and in certain cases to an illegiti- 

mate reorganisation of genetic material resulting in 

heightened cancer risk. 

A long-term replication or transcription stop causes 

the cell to destroy itself by means of a genetic programme 

called apoptosis (programmed cell death). Its place is 

taken by a neighbouring cell. As the neighbouring cells 

only perform a restricted number of divisions, the life ex- 

pectancy of the entire organism is reduced by the perma- 

nent stress caused by the absence of important control 

and repair systems. The Große lab investigates the con- 

nections between replication and transcription stalling, 

the triggering of signal pathways reporting these events 

to the cell cycle and repair machinery, and the responses 

with which cells protect themselves from mutations and 

chromosome rearrangements. 

Premature ageing 

When transcription is experimentally blocked, for ex- 

ample by administration of the inhibitor actinomycin D, 

the following processes are observable: Around the site 

of the damage triggered by actinomycin D a rapid change 

occurs affecting chromatin, the genetic material of the 

cell in the interim phase of cell division. Foci of a phos- 

phorylated form of the rare histone H2AX are formed. 

These foci mark the sites of the damage and direct fur- 

ther signal mediators and repair proteins to these places. 

Among the repair proteins there are two that are ca- 

pable of unwinding DNA: the helicases WRN and NDH II. 

We assume that NDH II removes incomplete transcripts,

whereas WRN prevents single-strand DNA from penetrat- 

ing transcription bubbles. This protects the cell from un- 

desirable recombination events. 

Humans without the repair protein WRN die prema- 

turely (no older than 54) and display all the features of 

ageing. Their hair goes grey, their skin is wrinkled, they 

develop cataracts and diabetes mellitus and are very likely 

to suffer from cancer. All these symptoms are known col- 

lectively as the “Werner syndrome”, named after the Kiel 

physician Otto Werner (1879-1936). 

How repair proteins cooperate 

As we have seen, the WRN helicase is important in 

preventing illegitimate recombination. But it also appears 

to perform many other functions as well. One of them is 

to watch over the integrity of chromosome ends (telo- 

meres), another to dismantle complicated DNA structures 

of the kind occurring in replication arrests. To this end, the 

WRN helicase cooperates with another enzyme, the DNA 

topoisomerase I. Accordingly, patients without the WRN 

helicase respond very sensitively to the administration of 

topoisomerase inhibitors of the kind used in cancer ther- 

apy. Normally, the topoisomerase removes the superheli- 

cal tensions from the DNA that invariably occur during 

replication and transcription. In addition, the topoisome- 

rase can “accidentally” induce recombination events that 

would otherwise be monitored and prevented by the 

WRN helicase. One of the aims of our research work is to 

achieve a better understanding of the way these two 

enzymes cooperate. 

A complex repair system 

assures that damage to the 

DNA is promptly removed 

and repaired. If this system 

fails, cells are prone to 

progress into cancer cells 

and/or to undergo premature 

ageing. 

Interestingly, topoisomerase I is strongly stimulated 

not only by the WRN helicase but also by the tumour sup- 

pressor protein p53. If p53 is not functioning properly, this 

results in the absence of an important control factor pre- 

serving the cell from uncontrolled divisions. Defective p53 

is to be found in about half of all human tumours. We 

have been able to demonstrate that via its amino acids 

47

48 

Große Lab 

302 to 320 p53 interacts with amino acids 156 to 170 of 

topoisomerase I and performs its stimulatory activity by 

way of this interaction. It enhances the DNA-relaxing ac- 

tivity of the topoisomerase and draws upon the recombi- 

natory activity of that enzyme. 

These findings allow the following tentative conclu- 

sion: Cancer cells contain high concentrations of defective 

p53. This protein can no longer prevent cells from dividing 

in an uncontrolled manner. However, it still interacts with 

topoisomerase and stimulates it to engender undesirable 

recombination events. This may explain why we frequently 

observe genomic instability in cancer cells. 

Undesirable arrangements 

Our investigations have shown that cells carrying out 

the cellular suicide programme (apoptosis) contain high 

concentrations of topoisomerase-DNA covalent com- 

plexes. Surprisingly, the formation of these complexes 

also occurs when the DNA is not damaged. Is this an at- 

tempt on the part of the cell to avoid programmed cell 

death? Or does the topoisomerase perhaps join forces 

with p53 and the WRN helicase to cause the DNA frag- 

mentation that precedes apoptosis? Another objective of 

our efforts is to elucidate these connections. 

Undesirable chromosome rearrangements favour the 

formation of cancer cells and probably also cell ageing. 

We hope that our investigations can contribute to a bet- 

ter understanding of the complex mechanisms involved, 

with a view to preventing the onset of cancer and other 

age-related diseases. We strongly interact, both within 

our scientific interests and on a methodological level, 

with the labs of Diekmann, Herrlich and Wang. 

Author: Frank Große 

Phone: 0049-3641-656291 

E-mail: fgrosse@fli-leibniz.de 

Left: TRF2, in red, lights up on 

the blue-coloured 

chromosomal DNA. 

Middle: The dividing cell is 

shown in phase contrast 

Right: Merge of both pictures 

Lab members: Frank Große, Anita Willitzer, Bernhard Schlott, Norma 

Baum, Marcel Kramer, Laura Steller, Irmgard Tiroke, Anja Rockstroh, 

Caroline Utermann-Kessler, Karl-Heinz Gührs, Annerose Schneider. 

Not pictured: Prasun Chakraborty, Sibyll Pollok, Suisheng Zhang

nuclear 

export signal 

dimerisation 

domain 

AUG1 

C H 

Zn 

C C 

AUG2 

1 2 3 

Trip6 

C H 

Zn 

C D/H/C 

Trip6 mRNA 

dimerisation 

domain 

C H 

Zn 

C C 

1 2 3 

nTrip6 

Focal adhesions Nucleus 

AAAAAA 

C H 

Zn 

C D/H/C 

An organiser protein with multiple interaction domains is 

made in two forms, the longer one helping cell attachment to 

a surface, the short one directing genes in the nucleus. 

Metastatic Migration: 

A Disastrous Property of Cancer Cells 

In the past decade, the focus of our work has been on 

the process of metastasis formation. We have been in- 

quiring how it is possible for tumour cells to leave their 

site of origin and settle at other locations in the body as 

metastases (secondary tumours). The outcome of our ini- 

tial investigations on this topic was the identification of 

proteins (markers) on the surface of tumour cells. One of 

these tumour cell markers, CD44v6, turned out to be a de- 

cisive helper (co-receptor) of the receptor tyrosine kinase 

“Met”. (V6 designates a protein segment which is only 

contained in a fraction of CD44 molecules. It is introduced 

into the protein by so-called alternative splicing.) 

Together with its helper, Met directs the movement of 

cells in the organism, e.g. during the development of the 

embryo, when cells – in accordance with the blueprint of 

the body – form organs and tissues and have to migrate in 

order to do this. Met also controls the migration of cells in 

the adult, e.g. immune-response cells. Not surprisingly, 

inappropriate cellular migration also depends on Met and 

its helper protein. Tumour-cell migration is responsible for 

incurable metastatic cancer. 

Herrlich Lab 

Tumour cells can detach from their site of origin, migrate to remote regions of the body and 

develop into secondary tumours or metastases. What it is that makes these cells capable of 

embarking on this fateful journey is a focus of the scientific work carried out at the Herrlich labora- 

tory. Peter Herrlich has described molecules that make a decisive contribution to the metastatic 

cascade. Hopefully, this knowledge may be of use in the development of interference strategies. 

How does the helper protein function? 

Our aim is to decipher the mechanism behind it. Ac- 

cording to our current knowledge, CD44v6, which pro- 

trudes from the cell membrane and reaches into the cell 

with a cytoplasmic tail, contributes to migration control 

in three ways. It is required for the activation of Met in 

response to its ligand, the so-called hepatocyte growth 

factor (HGF). This activation can be disturbed by the addi- 

tion of an antibody directed against v6 or of small pep- 

tide sequences corresponding to part of the v6 sequence. 

This may be a promising way of interfering with metas- 

tasis formation. Secondly, the cytoplasmic tail of CD44v6 

mediates the turn-on of the switch proteins Ras and Rac, 

a mechanism studied in conjunction with the Morrison 

laboratory. Since this mechanism involves the actin cy- 

toskeleton, which is also a target of viruses that cause hu- 

man cancer, we are attempting to identify intermediate 

components in this cancerous pathway (in collaboration 

with the Ploubidou laboratory). In a highly interesting 

third type of action, CD44 is cleaved to release the cyto- 

49

50 Herrlich Lab 

2. 

3. 

Cofactor for growth factor receptors 

Linkage to actin cytoskeleton 

HGF 

Met CD44v6 

ezrin 

1. Cell-cell and cell-matrix contact 

F-actin 

plasmic tail, which enters the nucleus and turns on a pro- 

gramme of gene expression. This programme promotes 

cell proliferation and cell migration. The cleavage resem- 

bles that of the Alzheimer precursor protein, so the mech- 

anism of CD44 cleavage may also tell us something about 

control steps in the production of Alzheimer plaques (we 

are collaborating on this with the Kaether laboratory). 

In the meantime, the co-receptor concept has proved 

to be valid for other receptors responsible for the recep- 

tion and mediation of growth messages, including the 

receptors Ron, Sea, Trk and PDGF. Ron, Sea and Trk need 

CD44v6 as a co-receptor, while the PDGF receptor is de- 

pendent on an integrin, beta-1. CD44 and the integrins 

are, in addition, adhesion molecules with which the cells 

make contact with other cells or connective tissue struc- 

tures. 

CD44, an artist among proteins: it reaches from the outside of 

the cell to the cell‘s interior, and it fulfils at least four 

functions: (i) mediating the contact of cells with its 

environment (the so-called extracellular matrix), (ii) helping 

several growth factor receptors, (iii) regulating through a link 

(by ezrin) to the actin cytoskeleton an important switch (see 

The Trip6 focal adhesion protein 

Several years ago the Herrlich lab and its collaborators 

discovered a chromatin-associated protein performing im- 

portant functions in gene regulation: Trip6. It does not 

bind to DNA directly but interacts with transcription fac- 

tors bound to its promoters. Through several specific do- 

mains Trip6 selects the transcription factor it binds to. 

Trip6 then appears to assemble various other regulatory 

factors required for the activation or repression of tran- 

scription. For instance, Trip6 enhances the transcription- 

activating function of AP-1 (Jun:Fos) and of NF-κB, whose 

actions promote cell survival, proliferation and migration. 

CD44 

full length 

Comet assay 

soluble CD44 

Ectodomain cleavage 

Intramembranous 

cleavage 

? ? ? ? 

CD44tail 

nucleus 

Morrison lab). Last but not least 

promoter 

CD44 is cleaved like the APP molecule, 

the precursor of the Alzheimer peptide. 

Cleavage releases the cytoplasmic tail which is transported to the 

nucleus where it promotes a gene program for migration and 

proliferation (right panel). 

Conversely, Trip6 mediates the repressive action of the 

gene 

expression 

glucocorticoid receptor on these transcription factors, a 

process studied in Jan Tuckermann’s laboratory. Interest- 

ingly, Trip6 is synthesized in two forms: a nuclear form, 

which we discovered, and a membrane-bound form, 

where it is associated with so-called focal contacts. The 

two forms are generated by alternative translation, i.e. 

the transcript carries two start signals for protein synthe- 

sis. Our current efforts are directed towards understand- 

ing the transcriptional function of nuclear Trip6 and iden- 

tifying the roles the two forms play in the mouse 

organism. 

Author: Peter Herrlich 

Phone: 0049-3641-656334 

E-mail: pherrlich@fli-leibniz.de

Homologous recombination, a rarely occuring DNA 

repair mechanism in human cells, can be visualised using 

appropriate reporter systems. 

Radiation sensitivity in tumour patients: 

the role of repair genes 

About five to ten percent of cancer patients undergo- 

ing radiotherapy respond so sensitively to radiation that 

the therapy causes adverse clinical symptoms. The aim of 

Eberhard Fritz, a member of the Herrlich laboratory, is to 

identify cellular and molecular markers with which the 

sensitivity of individual patients to radiation can be pre- 

dicted. Suspected causes of increased sensitivity to irradi- 

ation are, for example, genes or proteins involved in the 

repair of the DNA molecules. 

Together with our clinical partners, we are currently in- 

vestigating sensitivity to radiation using blood cells from 

cancer patients. For this purpose, we make use of so- 

called alkaline comet assays (see page 50) that measure 

the cells’ ability to repair DNA molecules previously dam- 

aged by experimental irradiation. In addition, we are also 

investigating how many blood cells are subject to so- 

called programmed cell death (apoptosis) after irradia- 

tion. A third parameter, radiation-induced phosphoryla- 

tion of the histone H2A protein, is an indicator of active 

DNA repair. Following such cellular characterisations, the 

subsequent aim is to search for molecular causes for in- 

creased radiation sensitivity in cells identified as “abnor- 

mal”. The starting point in this search is analysis of the 

genes and proteins already known to be involved in DNA 

repair. For example, our intention is to identify gene vari- 

ants – polymorphisms – that may be responsible for in- 

creased sensitivity to radiation. A further aim is to locate 

DNA repair proteins (green and red) attached to the DNA 

(blue) in chromatin or of condensed chromosomes are 

visualised by immunofluorescence techniques. 

genes, unknown until now, that mediate increased sensi- 

tivity to radiation. In doing so, we also draw on the exper- 

tise and technical resources of the Genome Analysis labo- 

ratory headed by Matthias Platzer. All our cellular and 

molecular experimental data on radiation sensitivity will 

subsequently be correlated with clinical data describing 

the course of the illness for each patient. 

Author: Eberhard Fritz 

Phone: 0049-3641-656371 

E-mail: efritz@fli-leibniz.de 

Lab members: Pavel Urbanek, Beate Voigt, Birgit Pavelka, Eberhard 

Fritz, Harald Seeberger, Juliane Rübsam, Monika Stopinska, Kristin 

Dreffke, Peter Herrlich, Kristin Platzeck. Not pictured: Tobias Sperka 

51

52 Heuer Lab 

Influential Messengers: 

How Thyroid Hormones Affect the Brain 

Without thyroid hormones the brain neither develops properly nor functions as it should. 

So far, however, we do not know how thyroid hormones actually get into the brain, nor how 

they manage to influence nerve cells. Heike Heuer and her team attempt to trace the molecular 

pathways on which thyroid hormones transmit their messages. 

Thyroid hormones are important signalling substances 

that affect basically every organ, most prominently the 

brain. Children born without a functional thyroid gland 

and not treated in time with thyroid hormones will even- 

tually develop a syndrome known as cretinism. These chil- 

dren will show severe mental impairments, suffer from 

hearing deficits and their fine motor functions will be dis- 

turbed. Neurological symptoms are also observed when 

thyroid hormone deficiency occurs later in life. But how 

do thyroid hormones make their way into the brain? 

Which processes inside the cells are regulated by thyroid 

hormones? And finally, how does ageing affect thyroid 

hormone functions in the brain? These are crucial ques- 

tions we aim to address in our studies in greater detail. 

In order to act in the brain, the prohormone thyroxine 

(T4) has to be produced by the thyroid gland and to be se- 

creted into the circulation. T4 is then taken up by the brain 

via the so-called blood-brain barrier and transported into 

glia cells that express a special enzyme called deiodinase 

type 2 (D2). This enzyme is capable of transforming T4 

into the active thyroid hormone T3. Subsequently, the glia 

cells release T3 that is then taken up by neurons. Finally, T3 

binds to its receptors (thyroid hormone receptors, TR) that 

are located in the nucleus and act as transcription factors 

thereby regulating the expression of respective target 

genes. 

Enigmatic transport pathways 

Accordingly, a number of transport processes are re- 

quired for thyroid hormones to be effective in the brain. 

At the molecular level, however, we do not know much 

about the proteins that are involved in these transport 

processes. One exception is the so-called monocarboxy- 

late transporter (MCT8), which was first characterised in 

2003 as an efficient, thyroid hormone-specific transporter 

(Friesema et al., 2003). 

The importance of this transporter became evident 

when patients were identified who carry mutations or de- 

letions in the gene coding for MCT8 that is located on the 

X chromosome. All patients with an inactive MCT8 trans- 

porter suffer from a severe form of psychomotor retarda- 

tion (also known as Allan-Herndon-Dudley syndrome), as- 

sociated with severe mental and physical disabilities

The effect of thyroid hormones on the brain can be 

demonstrated on Purkinje cells of the cerebellum. Right: 

Purkinje cells of normal mice. Left: Purkinje cells of mice born 

without a thyroid. 

(Friesema et al., 2004; Dumitrescu et al., 2004). In addi- 

tion, all patients exhibit very unusual serum thyroid hor- 

mone concentrations. This finding further supports the 

hypothesis that MCT8 also acts in vivo as an important 

thyroid hormone transporter. 

But why does the deactivation of the MCT8 trans- 

porter lead to such a severe disorder? In order to get 

insight into the pathogenic mechanisms we are 

currently studying genetically engineered mice 

mutants that are deficient in MCT8. Our pre- 

liminary analysis already revealed that 

MCT8 deficient mice show the same ab- 

normal thyroid hormone parameters as pa- 

tients with MCT8 mutations. As a conse- 

quence, all tissues analysed so far are affected 

with liver and kidney being in a hyperthyroid state 

while the brain was found to be in a hypothyroid condi- 

tion. Most importantly, by studying thyroid hormone 

transport processes we could demonstrate that in the ab- 

sence of MCT8 the uptake of the active thyroid hormone 

T3 into the brain is diminished. Based on these findings 

we speculate that an impaired uptake of thyroid hor- 

mones, especially during critical periods of brain develop- 

ment, might cause the severe neurological symptoms of 

the patients. Further studies are ongoing whether ageing 

also affects thyroid hormone transport processes in the 

brain and might therefore result in a decrease of cognitive 

capacities. 

“MCT8“ is the name of a transporter protein that takes thyroid 

hormones to the nerve cells. The corresponding gene is 

particularly active in the hippocampus, a region of the brain that 

is, among other things, responsible for learning and memory. 

Communication within neurons 

We are not only interested in elucidating the role of 

thyroid hormone transporters in the brain, but we also 

aim to define the signalling processes in neurons and glia 

cells that are controlled by thyroid hormones. Especially, 

we would like to understand by which 

pathways and proteins thyroid hor- 

mones regulate the development of 

the cerebellum, a key brain area in- 

volved in motor control. For that pur- 

pose we are studying the impact of 

thyroid hormones on Purkinje cells 

as the principle neuron of the cere- 

bellar cortex, and on Bergmann glia 

cells as an important neighbour support- 

ing Purkinje cell dendrite formation. By analysing 

hypothyroid mice we already identified Purkinje cell and 

Bergmann glia specific genes that are regulated by thy- 

roid hormones. In primary neuronal cell cultures and orga- 

notypic slice cultures we will further investigate the func- 

tion of these genes in promoting dendritogenesis, a 

process highly dependent on proper thyroid hormone sup- 

ply. These studies are linked to other FLI laboratories 

working on neurodegeneration, in particular to the labs 

Kaether and Wang. 

53

54 

Heuer Lab 

Hormone paths: The hormone T4 is produced by the 

thyroid; it must first overcome the blood-brain barrier and 

then enters the glia cells (supporting tissue) of the nervous 

system. Only the glia cells are able to convert the inactive 

hormone T4 to the active hormone T3. T3 then enters the 

neuron, presumably with the help of a transporter protein 

(MCT8). When it has reached its destination, T3 activates its 

receptor (TR), influences the transcription of genes and the 

production of special proteins by the cell. 

Thyroid hormones, neuropeptides and 

energy homeostasis 

Another focus of our research is to understand the 

mechanisms by which neuropeptides regulate body 

weight and food intake. By analysing mice mutants we 

are especially interested in studying the role of thyrotro- 

pin-releasing hormone (TRH) that not only functions as a 

hypothalamic-hypophysiotropic releasing factor thereby 

stimulating thyroid hormone production, but also acts as 

a neuropeptide in the CNS where it is involved in central 

circuits regulating energy homeostasis. This project prof- 

its methodologically from collaborations within the FLI 

“metabolic club” comprised of members from the 

Calkhoven, Bauer, Heuer and Tuckermann labs. 

Author: Heike Heuer 

Phone: 0049-3641-656021 

E-mail: hheuer@fli-leibniz.de 

Thyroid hormones stimulate the growth of dendrites, which 

are the short, heavily branched projections of a neuron. 

Lab members: Sigrun Horn, Katja Seider, Jan Lukas, Heike Heuer, 

Andrea Hirsch, Sabine Landmann. Not pictured: Marija Trajkovic

Alzheimer’s plaque: protein 

deposits with serious 

consequences 

… the most frequent form of 

dementia in Germany and 

most developed Western 

countries 

Misguided Proteins: 

Looking for the Causes of Alzheimer’s Disease 

Kaether Lab 

As society is getting older all the time, more and more people will come down with Alzheimer’s 

disease. Today, this disorder and the complete loss of personality that goes with it is the most 

frequent form of dementia in Germany. Christoph Kaether sums up what scientists know about the 

way the disease originates and explains the role played in its development by the misguided trans- 

port of proteins. The work being done by his research group in the general programme “age-related 

diseases” bears prospects for improved therapies. 

In 1906 the German neurologist Alois Alzheimer first 

described a syndrome involving a complete loss of per- 

sonality in persons suffering from it. At present, about 1.2 

million people in Germany are affected by Alzheimer’s dis- 

ease, which makes it the most frequent form of dementia 

in the country. Alongside the individual suffering this ill- 

ness causes its victims and their families, Alzheimer is also 

a major challenge for a society in which the progressive 

ageing of the population will further increase the number 

of patients. 

Protein aggregates 

Alzheimer’s disease involves the formation of abnor- 

mal protein deposits in the brain, so-called amyloid 

plaques and neurofibrillary tangles. The tangles consist of 

so-called tau proteins, while the amyloid plaques are 

largely made up of amyloid beta, small protein molecules 

consisting of 40 to 42 amino acids, the components of the 

proteins. 

Amyloid beta develops from a larger precursor mole- 

cule, the “amyloid precursor protein”, APP for short. This 

precursor molecule is anchored in the membrane surface 

of nerve cells. Enzymes called secretases progressively cut 

amyloid beta (Aß) out of this precursor molecule. Initially, 

the ß secretase cuts away the large N-terminal end of the 

molecule anchored in the cell. The result is a fragment 

that remains anchored in the membrane of the nerve cell 

(C99). C99 in its turn is cleft by the γ secretase. In this way 

Aß is released and can form deposits outside the cell. The 

second cleavage product (AICD) originating from this proc- 

ess makes its way into the interior of the cell. The alterna- 

tive process involves the α secretase. It cuts Aß in the mid- 

dle and thus prevents it from clumping to form Alzheimer 

plaque. Normally, these two degradative pathways are in 

a state of equilibrium. In Alzheimer’s disease one of the 

pathways (ß) may be upregulated and the other (α) down- 

regulated. 

55

56 Kaether Lab 

Alzheimer “families” 

The decisive step in the formation of amyloid beta is 

the processing (enzymatic transformation) of C99 by the γ 

secretase. This enzyme is a member of a recently charac- 

terised family of enzymes that share the same active cen- 

tre (a GXGD motif). The γ secretase is highly complex and 

is made up of four different proteins: presenilin (PS) 1 or 2, 

nicastrin (Nct), Aph1 and Pen2. Mutations of the PS genes 

can cause familial Alzheimer. So far, over 100 families with 

familial Alzheimer have been identified which carry a mu- 

tation of the PS 1 or 2 gene. The γ secretase processes not 

only APP but also a large number of other surface pro- 

teins. Of these the cell surface proteins “notch” is the one 

that has been most accurately characterised. 

At present our research work focuses on three major 

issues: 

1. axonal transport dysfunctions in Alzheimer’s disease 

(axons are the long extensions of nerve cells), 

2. the transport and processing of the “notch” cell sur- 

face protein in nerve cells, 

3. the assembly and transport of the γ secretase. 

γ secretase 

Presenilin Nicastrin Pen2 Aph1α/β 

Decisive step in the formation of Alzheimer’s plaque: Conversion of Presenilin, a protein in the membrane of neurons, by the 

enzyme complex γ secretase. 

Transport problems 

APP is transported via fast axonal transport. We are 

not entirely sure where the processing that forms amyloid 

beta actually takes place in nerve cells (neurons). Investi- 

gations undertaken so far suggest that in neurons of 

Alzheimer patients axonal transport is disturbed. We are 

directly studying axonal transport in living neurons. 

The cell surface protein “notch” is important for the 

development and maturation of the nervous system. For 

example, notch is required to ensure that the nerve cells 

of the developing brain are correctly wired up to one an- 

other. In the adult brain notch is an important factor in 

memory processes. 

Like APP, notch is a substrate of the γ secretase. We in- 

vestigate the transport and processing of notch in the in- 

terior of nerve cells. For this purpose we use nerve cells 

from embryonic rat brains grown on coverslips. Subse- 

quently the nerve cells are examined using microscopes, 

video microscopes and biochemical procedures. 

In certain aspects the γ secretase resembles the chan- 

nels in cell membranes through which ions are trans- 

ported. Both the γ secretase and the ion channels consist 

of various protein sub-units. In both cases only fully as- 

sembled complexes leave the endoplasmatic reticulum 

(ER), one of the cell’s synthesis and transport systems. By 

contrast, unassembled sub-units are retained or trans- 

ported back into the ER. This process is mediated by sig- 

nals called “ER retention signals”. We have been able to 

identify such ER retention signals in two sub-units of the

γ secretase. One of these signals is recognised by a protein 

called Rer1. At present we are attempting to clarify the 

molecular details of this newly discovered mechanism 

with a view to achieving a better understanding of Alz- 

heimer’s disease and improving its treatment. We have 

close collaborations with the labs of Heuer, Than, Wang 

and Große to get support in histology, protein expression 

and analysis, transgenic mice and others. We extensively 

use the FACS and mouse facilities and the microscope 

user group. 

Author: Christoph Kaether 

Phone: 0049-3641-656230 

E-mail: ckaether@fli-leibniz.de 

Neurons have the enormous task of 

transporting substances over large 

distances along their axons (the long 

projections arising from the cell body). 

Failures in this transport system can have 

dramatic consequences for the function and 

survival of neurons. 

The small images originate from a film 

showing the transport along the axon of a 

live neuron. The arrows point to transport 

units that move along the axon at a high 

speed. 

Lab members: Kerstin Hünniger, Slavomir Kacmar, Christina Valkova, 

Christoph Kaether, Daniela Reichenbach, Matthias Faßler 

57

58 Morrison Lab 

P Y 

Growth 

factor 

Growth 

factor 

receptor 

Y P 

Grb2 

Adhesion 

receptor 

SOS 

Ezrin 

Ras 

GDP 

Actin filaments 

Extracellular 

matrix 

How Switch Proteins Are Regulated to Control 

Proliferation and Neural Function 

G-proteins, their activation and function have been 

known for several decades. Recently, however, we discov- 

ered a novel step in the activation of small G-proteins like 

Ras and Rac. The activation requires ezrin, one of the 

ezrin-radixin-moesin-(ERM) family of proteins. For the ac- 

tivation of Ras and Rac, ezrin needs to link the cell’s mem- 

brane to the underlying actin cytoskeleton. The sites on 

the plasma membrane to which ERM proteins bind are co- 

receptors providing essential aid in the functioning of the 

receptor tyrosine kinases (RTK). Together with their co-re- 

ceptors, the RTKs’ job is to receive growth signals and 

transmit them to the cell´s interior. Co-receptors and re- 

ceptors are large transmembrane proteins that reach di- 

agonally through the cell membrane. The part of the co- 

receptor reaching into the inside of the cell encounters 

binding proteins such as the ERM proteins which, in turn, 

make contact with the actin filaments of the cell´s skele- 

ton (cytoskeleton). The most frequently encountered rep- 

resentative of these binding proteins is ezrin. 

When a growth stimulus reaches an RTK, a chain of 

signalling events is triggered in the cell´s interior. One of 

the important signalling steps involves the activation of a 

A cascade of signals started on the cells exterior and 

relayed into its interior is involved in proliferation control 

(see text for details). 

Cell membrane 

Ras 

GTP 

Raf 

MEK 

ERK 

Proliferation... 

In all cells a number of on-off switches are accomplished by the loading of small so-called 

G-proteins with GTP. In the active GTP-bound state the G-proteins affect numerous cellular 

processes. Helen Morrison is interested in the tight control of the activation state of the G-proteins 

and particularly in how this control is exerted in proliferation and the establishment and function 

of neural synapses. 

switch protein called Ras. We have found that the pres- 

ence of ezrin at the co-receptor in conjunction with the 

link to the actin cytoskeleton is necessary for the activa- 

tion of Ras. 

Exactly how ezrin catalyses the activation of Ras is 

currently being investigated at our lab in collaboration 

with the Herrlich laboratory. Ras shifts between two 

functional states. In the “on” state it is associated with 

GTP, an energy-rich nucleotide, while in the “off” state 

Ras is associated with the less energy-rich GDP. The on-off 

switch is influenced by regulatory proteins. When an RTK 

receives a growth stimulus, the protein called SOS, a 

guanine nucleotide exchange factor (GEF) triggers the 

“on” state, inducing a rapid exchange of GDP for GTP. 

What does ezrin contribute to this reaction? We have 

discovered that ezrin interacts directly with Ras-GDP 

(inactive Ras). In association with the actin cytoskeleton, 

ezrin also interacts with SOS. In the absence of ezrin, 

these interactions cannot occur and the formation of 

Ras-GTP is impossible despite stimulation of the RTK.

The acquisition of genetic changes (mutations) confers new 

characteristics to a cell facilitating it to leave its place of 

origin, move over to the blood or lymphatic system, and seed 

in a distant place forming metastasis. 

 

 

 

 

The idea we are following up is that ezrin associated 

with the actin cytoskeleton not only brings SOS and Ras 

together but also unfolds and activates SOS. Though this 

cascade of interdependent activation processes may 

sound confusing, it is in fact characteristic of highly im- 

portant control steps. SOS carries two Ras binding sites, 

only one of which performs the GDP-to-GTP exchange. 

The other binding site greatly enhances catalytic action 

but is blocked by so-called autoinhibitory domains in the 

the structure of SOS. At present we do not know how this 

inhibition process is triggered but we believe that this is 

where ezrin comes into play, enabling SOS to expose the 

critical Ras binding site necessary for effective catalytic 

interaction with Ras. Is this simply the dissection of “mo- 

lecular mechanics”? This exciting new step in Ras activa- 

tion is regulated in its turn. Ezrin has a counterplayer - 

merlin! (see page 12) 

Signal transduction in neurons 

So far, our work on the regulation of Ras and Ras-like 

proteins in physiological and pathophysiological situa- 

tions has concentrated on the development of cancer. The 

small G-protein Ras not only regulates essential cellular 

features such as proliferation but also controls other cellu- 

lar programmes such as cell death and differentiation. In 

addition, hyperactive Ras can induce senescence and cel- 

lular ageing, which, paradoxically, would help to avoid tu- 

mourigenesis. 

Our studies also focus on the conditions that influence 

the decision determining which Ras-dependent process is 

chosen. Here we plan to home in on the role of ERM and 

adhesion-dependent Ras regulation in the development 

 

 

 

 

 

 

 

Loss of contact inhibition of proliferation is one important 

feature in the malignant transformation process: the protein 

merlin indicates cellular contact and inhibits proliferation. 

Obstruction of merlin‘s function renders cells insensitive to 

contact signals leading to more proliferation and finally to 

metastasis. 

and function of neuronal synapses. It is well known that 

Ras and Ras-like proteins play a role in synaptic and struc- 

tural plasticity, the cellular and molecular basis for pro- 

cesses such as learning and memory. Synapses are dy- 

namic structures enabling nerve cells to communicate 

with one another through axons and dendrites, thus con- 

verting electrical impulses into chemical signals. The 

knowledge that CPI-17 is expressed in the brain has en- 

couraged us to inquire whether neurons make use of CPI- 

17 to control Ras activity through the interplay of MYPT-1/ 

PP1δ, merlin and ERM and, if so, which neurons are in- 

volved in this process. 

Author: Helen Morrison 

Phone: 0049-3641-656139 

E-mail: helen@fli-leibniz.de 

Lab members: Ingmar Scholl, Helen L. Morrison, Cui Yan, Sabine 

Reichert, Uta Petz, Katja Geißler, Ulrike Merkel 

59

60 Platzer Lab 

base pairs 

Genomes, Diseases and Ageing 

Cell 

histone 

nucleus 

chromatids 

chromosome 

DNA 

double strand 

For a better understanding of the way in which genes are involved in the origin of diseases and 

the process of ageing, scientists use intricate methods to investigate human chromosomes, 

compare the genomes of various organisms and draw upon new models, including a small fish 

During the international project devoted to the deci- 

phering of the human genome, our laboratory was in- 

volved in the analysis of the human chromosomes 8 and 

21 and the sex chromosome X. In the course of our work 

we were able to help in identifying and characterising 

genes responsible for human disorders, including short 

stature (SHOX), night blindness (CACNA1F), physical 

malformations (TRPS1), a skin disease (NEMO) and mental 

retardation (ATP6AP2), as well as a gene involved in DNA 

repair (NBS1). 

Remarkable chromosome 8 

Together with colleagues from the United States and 

Japan we discovered a remarkable characteristic of chro- 

mosome 8: a region of approximately 15 mb at the rear 

end of the so-called p-arm. Comparison of this chromo- 

some region of humans and chimpanzees reveals a major 

divergence. Also, the human population displays a much 

higher rate of polymorphisms (genetic differences within 

one population). This region contains a number of genes 

telomere 

centromere 

telomere 

among the shortest-lived vertebrates in existence. Matthias Platzer reports here on the research fin- 

dings and discusses genomic variations determining the individual risk for inflammation, infections, 

cancer, and adiposity, as well as ageing. 

significantly operative in the innate immunity system or 

the development of the nervous system. It is conceivable 

that these genes are subject to positive selection. 

Unique X chromosome 

Molecule of life: DNA 

and its organisation 

within the cell 

The biology of the X chromosome, the sex chromo- 

some shared by men and women, is unique, which is why 

geneticists have taken an interest in it from an early 

stage. Gene defects on the X chromosome mainly affect 

men because unlike women (= XX) men have only one X 

chromosome (= XY). The publication on the sequence of 

the human X chromosome that we were involved in indi- 

cates the value of international and systematically coordi- 

nated studies for the elucidation of fundamental biologi- 

cal and medical issues. The detailed analysis of the X 

chromosome revealed that, comparatively speaking, it is 

a chromosome with a relatively low number of genes. 

However, a disproportionately large number of hereditary 

diseases are associated with this sex chromosome. The 

remarkable thing is that about three-quarters of the

genes normally active in the male gonads and in tumours 

were found on the X chromosome. About 10 percent of 

the genes on the X chromosome are members of this 

“cancer-testis-antigen gene family”. This finding supports 

the hypothesis that in the course of evolution the genes 

providing the male with a selective advantage have been 

evolved especially quick on the X chromosome. But where 

this is associated with disadvantages for the female or- 

ganism their activity became restricted to testis. 

Comparative genome analysis 

Once the international “Dictyostelium discoideum Ge- 

nome Project” deciphering the genome of the social 

amoeba D. discoideum was completed, we began work on 

a comparison of the genomes of typical representatives of 

other groups of social amoebas. In this we have concen- 

trated on functional aspects. For example, we are at- 

tempting to identify “promoters” – those parts of a gene 

where transcription (the translation of DNA into RNA) be- 

gins – and are also characterising the telomeres, the “pro- 

tective caps” of the chromosomes. The telomere structure 

of D. discoideum was one of the big surprises produced by 

the genome project. It poses the question whether other 

social amoebas have developed mechanisms to maintain 

chromosome integrity. A second issue is whether this 

plays a role in the maturation and ageing of Dictyostelium 

cells. 

Detailed analysis of the human sex 

chromosomes (X,Y) shows that the Xchromosome 

belongs to the chromosomes 

that are rather poor in gene numbers. 

Quite revealing: Comparing the human chromosomes with 

the chromosomes of monkeys and apes. 

First disease gene for sarcoidosis 

In the field of functional genomics we collaborate 

closely with the Kiel campus of Schleswig-Holstein Uni- 

versity Hospital. A recent success this cooperation has 

come up with is the identification of a splicing-relevant 

polymorphism (rs2076530) in the BTNL2 gene. This gene 

variant is associated with increased risk of sarcoidosis, an 

ailment in which the immune system attacks tissues and 

organs in the body. Exchanging the base guanine for ade- 

nine in the last position of exon 5 activates a splicing do- 

nor four nucleotides further up the exon so that the alter- 

native splicing product displays a premature stop codon. 

As is usual in case for complex diseases, the individual 

sarcoidosis risk is only slightly increased. In the population 

as a whole, however, the probability of contracting this 

immune disease rose for the proband group by about 

23 percent. 

Fending off bacteria and cancer 

“Defensins” are proteins produced by cells to fend off 

bacteria and viruses. Defensins modulate cellular per- 

formance and in recent years they have been given 

greater attention by cancer researchers. The largest 

number of the defensin genes (DEF genes) are to be found 

on chromosome 8, more precisely in a 2-Mb locus at p23.1. 

We have elucidated the complex and dynamic structure of 

this locus. It is distinguished by extensive segmental du- 

plications and individual copy number variations. We have 

also been able to develop methods to accurately quantify 

61

62 Platzer Lab 

Human chromosomes; here: a female 

“karyotype” (XX) 

these structural polymorphisms and to show that the dip- 

loid copy number of a region as large as ~350 kb contain- 

ing eight DEF genes varies from 2-12. 

Experiments comparing the so-called haplotype pat- 

terns of patients with prostate cancer to those of healthy 

individuals indicated substantial differences between the 

two groups. Accordingly, we now intend to investigate the 

relation between such DEF haplotypes and DEF copies in 

cases of prostate cancer, notably with a view to providing 

a functional characterisation of predisposing haplotypes. 

Small causes, large protein diversity 

In the quest for genetic variations typically associated 

with complex diseases we have discovered a widespread, 

before largely neglected form of alternative splicing. 

“Splicing” is a process in which DNA sequences not coding 

for a protein (introns) are removed from the messenger 

molecule (mRNA). The remaining sequences (exons) are 

subsequently connected, thus producing an mRNA as a 

blueprint for protein synthesis at the ribosomes. In the 

case of alternative splicing the exons are connected in a 

different way, so that different proteins can be produced 

from one gene. 

In the so far neglected form of alternative splicing that 

we have described, two messenger molecules originate at 

each of the acceptor splicing sites with the sequence mo- 

tif NAGNAG. Some of these molecules contain the second 

NAG, others do not. NAGNAG or tandem acceptors have 

two acceptor AGs at distances of three nucleotides. This 

sequence motif frequently allows for the use of both AGs 

during the splicing process. Here the longer “E transcript” 

contains the second AG while the entire NAGNAG motif is 

intronic in the shorter “I transcript”. The introduction of a 

NAG into the coding sequence can either lead to the inclu- 

sion of a single amino acid or to the substitution of a com- 

pletely different dipeptide for one amino acid. A stop co- 

don can also be introduced. At the protein level this 

results in a wide range of variants, although this form of 

alternative splicing produces modified transcripts differ- 

ing only in three nucleotides. 

We have been able to show that NAGNAG acceptors 

are widespread both in the human genome and in others. 

About 30 percent of human RefSeq genes have at least 

one NAGNAG acceptor in the protein-coding sequence 

and experiments confirm that five percent have at least 

one functional NAGNAG. An interesting factor is that 

NAGNAG acceptors are not distributed randomly in the 

genome but display a number of significant features. Tan- 

dem acceptors demonstrate a marked inclination for in- 

trons in phase 1, lead much more frequently to the intro- 

duction/deletion of a single amino acid and result in polar 

amino acids enriching themselves on neighbouring exon 

boundaries. The proteins encoded by genes with NAGNAG 

acceptors interact very frequently with other proteins or 

nucleic acids. Depending on the use of the two acceptors, 

the form of alternative splicing we have described causes 

tissue-specific differences. In addition, the tandem accep- 

tors are conserved in the mouse and account for about

Alternative splicing 

at NAGNAG acceptors 

half of all alternative acceptors conserved. These facts 

substantiate the biological relevance of the subtle form of 

alternative splicing we have described. 

NAGNAG acceptors are frequently found in genes re- 

sponsible for the occurrence of diseases. For example, 

genes involved in adiposity or chronic inflammations of 

the intestines have corresponding tandem acceptors. 

AIDS takes different courses 

Together with scientists from the German Primate 

Centre in Göttingen and the Universities of Cologne and 

Kiel, we are investigating the most important animal 

model for the immune deficiency syndrome AIDS for host 

factors and genetic variability. These investigations are 

being conducted on related rhesus monkeys (Macaca mu- 

latta) infected with SIV (simian immune deficiency virus), 

in which the disease takes different courses. Re-sequenc- 

ing functional candidate genes has made it possible to 

identify a genetic variant that correlates with the variable 

course of the illness in rhesus monkeys. With the aid of a 

genome-wide mapping of the monkeys infected with SIV 

we are looking for other, hitherto unknown regions con- 

tributing to the inhibition of virus multiplication. Our fur- 

ther aim is to make a detailed study of candidate genes 

from this region and to add the knowledge thus acquired 

to the genomic characterisation of HIV patients displaying 

different illness courses. 

Massive-parallel sequencing 

(MPS) in picotiter plates 

(GS20, Roche) 

A fish model 

Lab members: Rica Zinsky, Andrew Heidel, Hella 

Ludewig, Stefan Taudien, Nadine Zeise, Patricia Möckel, 

Sabine Gallert, Niels Jahn, Cornelia Luge, Brigitte 

Küntzel, Bernd Senf, Silke Förste, Jeanette Kirschner, 

Tom Hofmann, Ivonne Heinze, Susanne Fabisch, 

Ivonne Görlich, Marie-Luise Schmidt, Ulrike Gausmann, 

Klaus Huse, Kathrin Reichwald, Karol Szafranski, 

Markus Schilhabel, Roman Siddiqui, Matthias Platzer. 

Not pictured: Chris Lauber, Marcel Kramer, Ralf 

Dittmann, Stefanie Schindler, Oliver Müller, 

Rileen Sinha, Marita Liebisch, Christin Heinrich, 

Nicole Ulbricht, Ulrike Sauermann, Daniela Werler, 

Christoph Sponholz, Beate Szafranski, Gernot Glöckner, 

Marco Groth 

The African turquoise killifish Nothobranchius furzeri is 

one of the most short-lived vertebrates in existence. It is a 

suitable experimental model for age research as it can 

conceivably be used to identify genes determining 

lifespan. In conjunction with the laboratories headed by 

Christoph Englert and Alessandro Cellerino we have initi- 

ated the first steps towards a N. furzeri genome project. 

Once completed it will provide the foundation for all fur- 

ther molecular, cell-biological and whole-organism inves- 

tigations providing insights into molecuar mechanisms of 

ageing and age-associated diseases. 

Author: Matthias Platzer 

Phone: 0049-3641-656241 

E-mail: mplatzer@fli-leibniz.de 

63

64 Ploubidou Lab 

Cytoskeletal signalling & transformation 

The cytoskeleton is made up of 3 types of filaments: 

actin microfilaments, microtubules and intermediate fila- 

ments. These filaments form highly organised structures 

(see figures) that have structural functions in the cell, are 

required for cell motility and are modulated through in- 

tra- and extracellular signals which are important for 

overall cellular function and tissue homeostasis. In fulfill- 

ing these functions, the cytoskeleton acts as a signalling 

center, converting intra- and extra-cellular signals into 

structures and structure remodeling. This diversity of cy- 

toskeletal functions is reflected in the use and abuse of 

the cytoskeleton in disease. 

Pathogens subvert cellular processes to their own ad- 

vantage and a common recurring target is the cytoskele- 

ton. Thus they provide efficient, manipulable systems to 

dissect cytoskeletal function, its regulation and the cross- 

talk among cytoskeletal components. We are using vac- 

The actin cytoskeleton. Actin 

filaments are shown in yellow 

and the nucleus in blue. 

Virus-Induced Signal Transduction and Oncogenesis 

Cancer is a major age-related pathology, its incidence exponentially increasing after the age of 

50. Ageing is thus the largest single risk factor in the development of cancer. This is consistent 

with the fact that accumulation of genetic and epigenetic changes contribute largely to tumouri- 

genesis. Research in Aspasia Ploubidou‘s laboratory investigates how viruses modify cytoskeletal 

signalling, leading to oncogenic transformation and cancer. 

cinia virus in such an approach, to dissect centrosome and 

microtubule remodeling pathways and their dysregula- 

tion. The emphasis of our work is on signalling molecules 

which act as switches for the organisation and function of 

the cytoskeleton. 

Virus-induced oncogenesis 

The microtubule cytoskeleton. 

Microtubules are shown in blue 

and the nucleus in red. 

Approximately 20% of all cancers are virally induced. 

Our hypothesis is that virus-induced cytoskeletal transfor- 

mation plays a central role in infection-mediated onco- 

genesis. We are using human papillomavirus (HPV) as a 

model system for these studies. There are over 100 

human papillomaviruses classified as “low-” or “high-risk” 

in accordance with their pathogenic potential, which cov- 

ers a range extending from low-grade epithelial lesions 

exhibiting cytological abnormalities to skin papillomas 

(benign tumours) to highly invasive carcinomas.

We are studying the molecular basis of cytoskeletal 

function and dysfunction in HPV-induced malignant 

transformation, by dissecting the signalling pathways tar- 

geted early in transformation and by defining the struc- 

tural basis of the identified protein-protein interactions 

(collaboration with Görlach lab). We are using a cell cul- 

ture system and relate the obtained results to molecular 

and cellular changes that we observe in different stages 

and types of human cancer. 

Four different approaches are employed: Expression 

of the oncoproteins E6 and E7 from high-risk and low-risk 

HPVs in a cell culture system allows analysis of cytoskele- 

tal reorganisation in their cancer-related cellular traits and 

facilitates comparison with other oncogenes, such as the 

E1A adenoviral oncoprotein (collaboration with Herrlich 

lab). In a second approach, cells expressing affinity 

tagged E6 of high-risk and low-risk HPVs are used to ana- 

lyse the differences of protein-protein interactions among 

the different E6 oncoproteins with cytoskeletal molecules, 

by quantitative MS (collaboration with Große lab). These 

methods are complemented via the analysis of HPV-posi- 

tive tumours from patients at early and advanced cancer 

stages. As common cellular targets of oncogenic viruses 

are emerging, a systematic comparative study of viral on- 

cogenes is an efficient way to identify consensus host 

pathways manipulated by different oncoproteins. In a 4th 

approach, automated high-throughput technologies (HCS 

microscopy, chemical compound & RNAi libraries) that 

quantify cancer-related cellular traits are used to identify 

The microtubule cytoskeleton. 

Microtubules are shown in green 

and the nucleus in purple. 

new molecules, implicated in different stages of cancer 

progression (collaboration with Morrison, Kaether, Tucker- 

mann and Herrlich labs). 

Author: Aspasia Ploubidou 

Phone: 0049-3641-656468 

E-mail: ploubidou@fli-leibniz.de 

The cytoskeleton during mitosis. 

Microtubules are shown in 

green, actin in red and the 

nucleus in blue. 

Human papilloma virus 16: 

L1 capsid model, 

after Modis et al. (2002) 

Lab members: Dirk Schudde, Katja Bierhals, Jana Hamann, 

Juliane Simon, Yu-Chieh Lin, Aspasia Ploubidou, David Schmidt 

65

66 Schilling Lab 

Molecular Mechanisms of 

Huntington’s disease (HD) is an autosomal dominant, 

progressive and fatal neurodegenerative disease that usu- 

ally starts in mid life. It occurs when the nucleotide repeat 

sequence CAG (encoding glutamine Q) near the N-termi- 

nus of the huntingtin (htt) protein expands to a length 

greater than 36 consecutive glutamines. The symptoms of 

Huntington’s disease include motility disorder, cognitive 

impairment and psychiatric disturbances that lead to 

death after a period of 15-25 years. The neuropathological 

features of the disease include general brain atrophy and 

a dramatic loss of medium spiny neurons. 

In an earlier study we generated HD transgenic mice. 

These mice express the first 171 amino acids of the hunt- 

ingtin (htt) protein, including glutamine stretches of dif- 

ferent lengths, either 18Q, 44Q or 82Q (HD-N171-82Q). 

Expression is controlled by the mouse prion protein pro- 

motor. Only the 82Q mice display loss of motoric function, 

abbreviated lifespans and widespread nuclear and cyto- 

plasmic aggregates of mutant htt protein in the brain. We 

are attempting to establish the way in which proteolytic 

processing of the htt protein may be important in HD and 

could hence serve as a target for therapy. 

Proteolytic processing of htt 

Various studies have demonstrated that proteolysis of 

the htt protein is implicated in the pathogenesis of Hunt- 

ington’s disease. For example, neurons throughout the 

central nervous system harbour inclusion bodies in both 

the nucleus and cytoplasm that are immuno-reactive with 

antibodies directed against the N-terminal regions of htt 

but not against the C-terminal regions. N-terminal frag- 

ments of mutant htt proteins were identified in immuno- 

blots of homogenates from HD brains and transgenic 

mouse models. 

Map of the human huntingtin (htt) 

protein showing the localisation of 

the polyclonal peptide rabbit 

antibodies generated against 

several epitopes within the N171 

transgenic protein. 

Huntington’s Disease and Therapeutic Approaches 

The genetic cause of Huntington’s disease (chorea Huntington) was discovered back in the 

1990s. But the molecular mechanisms leading to the accumulation of protein fragments and 

the reasons for their toxicity in brain cells are still unknown. Gabriele Schilling describes how small 

fragments of the huntingtin protein can be generated by specific cleavage and explains why these 

fragments are related to, or possibly even cause, the pathogenesis of Alzheimer’s disease. 

In cell-culture models, short N-terminal fragments of 

mutant huntingtin protein are more toxic than full-length 

mutant htt proteins. Importantly, transgenic mice ex-

pressing only N-terminal portions of mutant htt protein 

develop pathological abnormalities that are nearly identi- 

cal to those found in humans. Accordingly, there is strong 

evidence suggesting that truncation of mutant htt pro- 

tein may play a role in creating a toxic, or toxic and aggre- 

gating, protein. 

Epitopes present in nuclear inclusions found in 

post-mortem HD brains. 

We had previously demonstrated that expression of 

N-terminal fragments of huntingtin ending at residue 171 

with stretches of 18, 44 or 82 glutamines, results in accu- 

mulation of htt proteins in the brain. These proteins com- 

prised both a fragment of the predicted size and a shorter 

C-terminally truncated fragment. We generated a panel of 

antibodies targeting several sequences in the N-terminus 

of huntingtin protein for use in immuno-cytochemical 

studies (see figure on page 66) of HD-N171-82Q mice and 

post-mortem HD brains. Our goal was to further charac- 

terise the mutant human huntingtin fragments that accu- 

mulate in inclusions found in human HD and in our N171- 

82Q mouse model of HD. We have been able to show that 

the huntingtin fragments making up nuclear inclusions in 

the brains of HD patients (see figure, this page) and N171- 

82Q mice possess C-termini that end between antibody 

epitopes defined by two htt peptides (81-90 and 115-129). 

Using biochemical tools, we now hope to identify the 

exact cleavage site of htt by expressing a fragment com- 

prising roughly the first 100 amino acids (Cp-A). In addi- 

tion, we have identified interesting mutations in C-pA dis- 

playing reduced or increased accumulation of the 

fragments in cells. Our next efforts will focus on determin- 

ing the toxicity of these Cp-A mutants by investigating the 

aggregation and the nuclear localization of the mutants. 

We also intend to continue with the search for pro- 

tease inhibitors in our cell-culture system, which has pro- 

duced promising results so far. If we can reduce htt cleav- 

age, we may be able to prevent nuclear localization and 

aggregation of mutant htt, which we believe may cause 

toxicity in Huntington’s disease. 

Microtubule disruption in HD 

Htt has been shown to interact with a variety of pro- 

teins linked to microtubule transport, including endophilin 

A, kinesin light chain and dynactin p150Glued via HAP-1. 

It has been suggested that these proteins function to- 

gether with htt protein in the retrograde axonal transport 

of organelles along the microtubules. In cooperation with 

A. Ploubidou, we aim to determine the precise role of mi- 

crotubule transport and the extent of microtubule dys- 

function in the brains of our HD transgenic mice. 

Author: Gabriele Schilling 

Phone: 0049-3641-656042 

E-mail: schillling@fli-leibniz.de 

Lab members: Christina Weiße, Stefanie Sendelbach, Gabriele 

Schilling, Katrin Jünemann, Denise Reichmann 

67

68 Sühnel Lab 

From Information to Knowledge: 

New Databases and Analysis Tools 

In the last decade no other branch of science has equalled modern biomolecular research in producing 

colossal amounts of data requiring collection, storage, validation, analysis and interpretation. 

Bioinformatics is a new scientific discipline designed to assist in coping with this data deluge. 

Jürgen Sühnel describes new databases and tools developed in Jena to getting the most out of data 

relating to biological sequences and three-dimensional structures. 

In the last decade, biology has probably been the sci- 

ence that has produced the largest amount of new scien- 

tific data at the quickest rate. This data explosion has led 

to the emergence of a new discipline, bioinformatics. The 

effects of this glut of new data are here to stay and they 

may well institute dramatic changes to the very approach 

to biological research. With the methods it has devised, 

bioinformatics is an operative factor in gradually taking 

this branch of science back outside the frontiers of reduc- 

tionism and towards a holistic, systemic and integrative 

approach to biological research. 

Bioinformatics has paved the way for this new per- 

spective by developing and supplying databases with 

which the plethora of new data can be captured, stored, 

tested and made available to users for analysis and inter- 

pretation. Largely speaking, modern biological research 

would be inconceivable without databases of this nature. 

But there are problems involved with these new sources 

of information. Users find it increasingly difficult to locate 

the data that are of interest to them. Here, database pro- 

ducers can improve matters by devising methods for 

greater data integration. A second problem has to do with 

the fact that many data resources have been developed 

for bioinformatics specialists, so that little attention has 

been paid to ensuring that they can be used intuitively 

without expert knowledge of informatics or computers. 

One of the key concerns in our work over the past few 

years has been to develop data resources and analytic 

tools with which the problems outlined above can be 

avoided. Two of these resources are described in the fol- 

lowing. 

The Jena Library of Biological Macromolecules, JenLib 

for short (www.fli-leibniz.de/IMAGE.html), serves to en- 

hance access to information on the three-dimensional 

structures of biological macromolecules. The software and 

database system GenColors (www.gencolors.fli-leibniz.de) 

is the basis for a series of browsers for prokaryotic ge- 

nomes. 

Three-dimensional 

DNA tetraplex 

structure: It is part 

of the telomeres, 

the „protective 

caps“ of the 

chromosomes.

Structure of a subunit of the ribosome (the place where proteins 

are produced) of the bacterium Haloarcula marismortui. 

The JenaLib database has been in existence since 1993. 

Starting in 2005 it has been substantially expanded in the 

framework of a project run by the National Genome Re- 

search Network. The first genome browsers based on 

GenColors were released in 2005. They developed in the 

course of a joint project conducted by the Jena Bioinfor- 

matics Centre in conjunction with the “Genome Analysis” 

lab headed by Matthias Platzer. 

“JenaLib”: Three-dimensional structures of 

proteins and nucleic acids 

The Jena Library of Biological Macromolecules uses 

structural information from primary data sources on 

three-dimensional structural data for biological macro- 

molecules, the Protein Data Base (PDB) and the Nucleic 

Acids Database (NDB). In addition, it makes numerous an- 

alytic methods of its own available to users and places 

special emphasis on data integration. The value added to 

the original data in this way is considerable. The JenaLib 

database consists of two parts. On the one hand it pro- 

vides general information on the architecture of biological 

macromolecules, on the other it contains a structural at- 

las with currently about 50,000 entries. With the aid of a 

specially designed system of cross-references UniProt pro- 

tein sequences and sequences extracted from structural 

files can be related to one another. In this way informa- 

tion hitherto only available on the sequence plane can be 

mapped onto three-dimensional structures. We have ap- 

plied this mapping technique to SAPs (single amino acid 

polymorphisms) and PROSITE motifs (PROSITE is a data- 

Structure of the protein Leptin. 

It plays an important role in 

regulating the lipid 

metabolism. 

base for functionally significant sequential motifs) as well 

as to the domain organisation of proteins provided by the 

Pfam database and to exon-exon boundaries. Recently, 

we have integrated information from the GenAge data- 

base that offers data on genes related to ageing. 

Visualisation is especially important for three-dimen- 

sional structures. Recently we developed the JenaLib 

Viewer that is based on the open-source Jmol Viewer 

(jmol.sourceforge.net) and offers a user-friendly and flex- 

ible Javascript interface. Unlike other visualisation tools 

the JenaLib-Jmol Viewer is platform-independent and en- 

ables the user to analyse biological macromolecules in a 

wide variety of ways with one and the same tool. One op- 

tion is the simple, alternative display of biological and 

asymmetrical units and standard visualisations of 

PROSITE, SCOP, CATH and SAP data. 

Flexible search options enable the user to home in on 

selected structures. JenaLib contains links to over 30 other 

databases and is quoted as a cross-reference by databases 

like SwissProt, OCA and PDBsum. The JenaLib database 

has already been reviewed twice by the renowned scien- 

tific journal Science. Images from the database have been 

widely made use of in books and exhibitions. In addition, 

the journal RNA (www.rnajournal.org) has used molecule 

images from JenaLib for its cover pages since 2003. The 

combination of available analysis tools and effective data 

integration makes the JenaLib database a unique resource 

for three-dimensional structures of biological macromole- 

cules. 

69

70 Sühnel Lab 

Images of the „Jena Library“ can be found on the 

covers of international scientific journals. Right: 

Circular representation of the hereditary material of 

the bacterium Escherichia coli, generated with the 

„Jena Prokaryotic Genome Viewer“. 

“GenColors”: Analysis of prokaryotic 

genomes made easy 

GenColors is a new software and database system 

that can be accessed via the internet or installed locally. 

The system makes the analysis of the genomes of 

prokaryotes (unicellular organisms, e.g. bacteria, whose 

cell nucleus is not membrane-bound) both better and 

faster. Genome comparisons are heavily drawn upon in 

the process. The system provides for the seamless incor- 

poration of data from ongoing genome projects into ge- 

nomes that are already complete. Export and import fil- 

ters facilitate simple data exchange, both with assembly 

programmes like GAP4 and with genomic data in Gen- 

Bank format. Most comparative genomics methods rest 

on the identification of so-called best bi-directional hits 

for protein sequences. These are used for the analysis of 

gene sequences, syntenies and gene core units in two or 

more genomes. Precalculated UniProt hits for all protein- 

coding genes make for effective annotation. To the extent 

that they are available, base-specific quality data (confi- 

dence, coverage) can also be processed. The GenColors 

system can be used both for annotation in ongoing ge- 

nome projects and as a tool for the analysis and presenta- 

tion of genomic data pertaining to complete genomes. 

The genome browsers based on GenColors come in 

two varieties, either as so-called dedicated browsers or in 

the form of the Jena Prokaryotic Genome Viewer (JPGV). 

Dedicated genome browsers contain information on a set 

of related genomes and provide a complete range of op- 

tions for genome comparison. The system was employed 

in the sequencing of Borrelia garinii, a European species 

causing borreliosis, and is at present being used in ongoing 

genome projects related to strains of Borrelia, Legionella, 

Escherichia and Pseudomomas. One of these dedicated 

browsers, the Spirochetes Genome Browser SGB (sgb.fli- 

leibniz.de) featuring Borrelia, Leptospira and Treponema 

genomes, is already freely accessible.

Unlike the dedicated browsers, the Jena Prokaryotic 

Genome Viewer (jpgv.fli-leibniz.de) contains information 

on all currently known complete bacterial genomes. At 

present, the JPGV contains some 1,140 genomic elements 

(chromosomes, plasmids) for 293 species. 

Alongside the functions outlined so far, both the dedi- 

cated browsers and the JPGV boast flexible and effective 

quick-search and advanced-search options and provide 

various possibilities for the generation of linear and circu- 

lar genome topologies. Predictions on horizontal gene 

transfer are the latest development to be incorporated. 

Future work will focus on improving the analysis of inter- 

genic regions and the search for small functional RNA 

molecules. 

Lab members: Eberhard Schmitt, Rolf Hühne, Marius Felder, 

Jürgen Sühnel, Friedrich Haubensak, Gerhard Müller, David Krahmer. 

Not pictured: Kristina Mehliß 

Author: Jürgen Sühnel 

Phone: 0049-3641-656200 

E-mail: jsuehnel@fli-leibniz.de 

71

72 Than Lab 

From Structure to Function: 

How Proteins Work in the Body 

In order to truly understand the function of proteins and biomolecules, as well as their interaction 

with other molecules at the atomic level, it is essential to have a detailed knowledge of their threedimensional 

structures. We employ the complex method of protein crystallography to determine the 

structures of proteins crucial for the ageing process and for age-associated diseases, making use of 

the diffraction of X-rays on the lattice structure of protein crystals. Manuel Than, head of the pro- 

tein crystallography laboratory, gives examples of applications and explains the importance of the 

method, which is also an essential basis for the target-oriented development of drugs. 

We explore the detailed three-dimensional structure 

of proteins in order to understand their function and their 

interactions during life, development and ageing at the 

atomic level. Such studies are also essential for the ra- 

tional development of organic compounds that influence 

protein activities and thus represent a basis for drugs tar- 

geting diseases that are difficult to treat or have even 

been incurable to date. Our laboratory uses a combi- 

nation of protein crystallography and biochemical 

and biophysical methods to analyse the structure 

and its impact on the function of proteins involved 

in the development of Alzheimer’s dis- 

ease and of proteolytic enzymes trans- 

forming inactive pro-forms of proteins into 

their active, mature form. This activation 

process is indispensable for the cellular gen- 

eration of a large number of proteins and 

factors required for cancer progression and 

metastasis but it also activates bacterial and 

viral pathogens. 

Structure and function of proteins related 

to Alzheimer’s disease 

Alzheimer’s disease is the most common form of de- 

mentia worldwide. In the brains of affected patients, pro- 

teins aggregate to form plaques, mainly consisting of so- 

called amyloid β-peptides (Aβ).These peptides are 

generated when a larger precursor protein, the β-amyloid 

precursor protein (APP), is cleaved by a specific set of pro- 

teolytic enzymes. Of special importance is the cleavage 

by γ-secretase, a membrane-integral enzyme complex 

of high molecular weight, which results in the liberation 

of Aβ by a chemical cleavage mechanism that can not be 

understood by known protease structures. In addition, a 

second cleavage product (AICD) is released, which is be- 

lieved to play a central role in cellular signal transduction 

(see also Kaether lab, page 55). In recent years, cell biolog- 

ical and biochemical research has provided a tremendous 

number of new insights into the pathological processes 

that eventually lead to the formation of Aβ. However, little

An enzyme gets in contact with its inhibitor. 

is known so far about the detailed atomic structures of the 

molecules involved, their interactions and the functions 

that these proteins normally fulfil in the healthy organism. 

The desire to learn more about these proteins, their struc- 

ture and function is the motivation behind many of our re- 

search projects, which are embedded in our institute’s re- 

search activities on Alzheimer’s disease. 

Activation of protein precursors 

Cells in an organism produce numerous proteins re- 

leased into their surroundings by the secretion machinery 

of the cell. Many of these secreted proteins are initially 

produced by the cell as inactive pro-proteins. The neces- 

sary activation takes place in the secretory pathway, just 

before their release from the cell, and is carried out by 

certain proteolytic enzymes constituting an enzyme fam- 

ily of their own, the so-called proprotein/prohormone 

convertases (PCs). Proteolytic cleavage by the PCs occurs 

in a calcium-dependent manner and only at very specific 

sites within the substrates. Proteins activated in this way 

include the blood-sugar regulating hormone insulin, vari- 

ous growth and differentiation factors and extra-cellular 

proteolytic enzymes. The latter are believed to play a cru- 

cial role in the development of neurodegenerative dis- 

eases, in oncogenesis and in metastasis. Also certain bac- 

terial toxins and viral coat proteins have to be activated in 

this way to become pathogenic. As they activate a large 

number of pathogenic proteins, the PCs constitute a very 

interesting pharmacological target. 

During the last few years we have been able to deter- 

mine the three-dimensional structure of the biochemically 

best characterised member of the entire PC-family, furin, 

Protein crystals are the basis 

for x-ray crystallography. 

by protein crystallography. Moreover, modelling studies 

have led us to propose very accurate structures of other 

family members as well. These studies enabled us for the 

first time to analyse the exact architecture of the PCs, to 

understand the substrate specificity of furin and of its 

close homologues and to develop a clear defined struc- 

tural concept of the activation of the PCs. Our next aim is 

to explore the structure of other family members, some 

of which differ considerably from furin in that they recog- 

nise different substrate sequences for cleavage. In addi- 

tion, we would like to advance the rational, structure- 

based development of inhibitors. 

Methodological improvements 

Our main goal is to use our protein-crystallographic 

expertise to investigate and understand the structure of 

our target proteins. However, the specific issues posed by 

a given project often necessitate the adaptation of exist- 

ing methods or the development of new ones. One of our 

latest developments is a special electron density map 

that made it possible to precisely identify the number 

and spatial location of calcium ions within the furin mol- 

ecule. Moreover, several projects have benefited greatly 

from a special treatment of protein crystals. Using spe- 

cific and tightly controlled changes of the humidity in the 

crystal environment we were able to improve the internal 

order of the protein crystals and thus facilitate the deter- 

mination of their structures. 

Author: Manuel Than 

Phone: 0049-3641-656170 

E-mail: than@fli-leibniz.de 

Lab members: Dietmar Schwertner, 

Miriam Küster, Sven Dahms, Janine Roy, 

Dirk Röser, Yvonne Schaub, Manuel Than 

73

74 Tuckermann Lab 

Steroid Hormones: 

Regulators of Tissue Integrity, Metabolism and 

Inflammation 

Steroids, in particular glucocorticoids (GCs) are widely 

used to treat allergic and autoimmune diseases due to 

their unsurpassed anti-inflammatory efficacy. However, 

their application is accompanied by severe side effects 

such as insulin resistance, fat redistribution, muscle and 

skin atrophy and osteoporosis. 

GC actions are exerted through the GC receptor (GR) 

(Figure 1). This nuclear receptor resides in the cytoplasm 

under resting conditions. After hormone binding the GR 

translocates into the nucleus where it alters gene expres- 

sion by acting as a transcription factor via several modes 

of actions. Those include the binding of a dimerised GR 

Fig. 1: The glucocorticoid receptor (GR) controls regulation of genes in two major 

ways: Two receptor molecules team up to a pair, a dimer, and bind to the DNA. 

The second mechanism is the interaction of a single GR molecule with other DNA 

bound transcription factors controlling inflammation (AP-1, NF-kB and STAT5). 

Steroids e.g. glucocorticoids are frequently used compounds to treat chronic inflammatory 

disorders and are involved in the treatment of certain cancers. The lab of Jan Tuckermann aims 

to get insight into physiological processes that are targets of the therapeutic effects and side 

effects of steroid hormones. We use for this purpose cell type and function selective gene targeted 

mice for the estrogen receptor, the glucocorticoid receptor and interacting factors such as STAT5. 

From these studies we could make significant contributions to the understanding of cellular and 

molecular action of steroids in contact allergy, in the septic shock response, glucocorticoid induced 

osteoporosis and in the contribution of the niche of hematopoeietic stem cells. 

molecule to palindromic elements in the promoter of GC- 

regulated genes and the interaction of the monomeric re- 

ceptor with DNA-bound transcription factors such as NF- 

kB, AP-1, IRF-3, and STAT5. Currently, suppression of those 

transcription factors is believed to underlie at least in part 

the anti-inflammatory effects of GCs, whilst dimerisation 

of the GR was hypothesised to contribute to the majority 

of side effects. 

In order to test this hypothesis we are employing func- 

tion selective GR mutant mice which carry a GR that is not 

able to dimerise and bind to palindromic elements, but re- 

tains its capacity to interact with pro-inflammatory tran-

wt 

GR dim 

CD68 

A 

Ox Ox + Dex 

CD3 

DAPI 

B 

C D 

scription factors (Fig. 2). Using these mice we have a pow- 

erful tool in hand to measure the contribution of the 

dimerisation in therapeutical and side effects of GC ther- 

apy. We employed these mice to determine the impact of 

dimerisation of the GR versus monomeric functions in 

contact allergy, sepsis and osteoporosis. These experi- 

ments were complemented by cell type specific deletions 

of the GR gene in mice to identify the relevant cell type 

mediating the GC action in vivo. This allowed us to investi- 

gate primary actions of the GR ex vivo on activation or 

differentiation and on the identification of dimer-depend- 

ent or dimer-independent down stream factors. 

The role of the GR in 

inflammation – Contact hypersensitivity 

Contact dermatitis is a common allergic 

reaction of the skin in which usually 

glucocorticoids (GCs) are prescribed 

as standard therapy. We could show, 

that in contact allergy anti-inflam- 

matory action of GCs is dispensable 

in keratinocytes, T-cells and antigen 

presenting cells, but absolutely required 

in macrophages and neutrophils to prevent 

CHS. Suppression of contact allergy by GCs requires the 

dimerisation of the glucocorticoid receptor (GR), since 

mice with a dimerisation defective GR (GR dim ) were largely 

resistant to GC treatment. 

Septic shock 

Sepsis is viewed as a complex dysregulation of inflam- 

mation arising when the host is unable to successfully de- 

Fig 2: During contact allergy, elicited by agents 

such as oxazolone (ox) leukocytes, in particular 

CD68 positive macrophages (green fluorescence) 

immigrate into the inflamed tissue in wild type 

(wt) and mice with a GR deficient in dimerisation 

(C, GRdim[superscript]). Glucocorticoid treatment 

(Ox + Dex) suppress potently the immigration of 

macrophages in wild type animals (B), but not in 

animals with a glucocorticoid receptor defective 

in dimerisation (D). 

feat an infection. We could show that for resolution of 

lethal inflammation in septic shock the dimerisation 

function of the GR in macrophages is required, since mice 

with the GR dim mutation in the hematopoeitic system and 

lacking the GR in macrophages (GR LysMCre ) exhibit a higher 

lethality to sepsis. The enhanced lethal symptoms in LPS 

treated GR dim and GR LysMCre mice were associated with a 

prolonged drop of glucose levels, whereas in wild type 

mice the glucose levels after an initial drop could be sta- 

bilised. This was a consequence of an impaired suppres- 

sion of IL-1b in the serum by endogenous GCs. Using re- 

combinant IL-1 receptor antagonist we could rescue the 

drop of glucose levels in GR dim and GR LysMCre mice. 

Glucocorticoid induced osteoporosis 

Osteoporosis is a severe adverse effect of 

glucocorticoid (GC) therapy. To explore 

the cell autonomous role of the GR in 

osteoblasts, we generated mice with a 

deletion of the GR in osteoblasts 

(GR Runx2Cre ). Treatment of GR Runx2Cre with 

GCs renders them completely resistant 

against GC induced bone loss and impair- 

ment of bone formation. In contrast, mice car- 

rying a DNA binding defective GR (GR dim ) responded to 

prednisolone treatment with an effective inhibition of 

bone formation leading to bone loss. These results indi- 

cate that selective GR agonists designed for non-dimeri- 

zation of the GR might not be suitable to avoid GC in- 

duced osteoporosis as a side effect of steroid therapy. 

75

76 Tuckermann Lab 

A B 

C D 

Fig 3: Macrophages isolated from wild type mice (A) react 

strongly to glucocorticoid treatment and reduce their 

inflammatory activity (B). Macrophages from mice with a 

dimerisation defective glucocorticoid receptor (C) are resistant to 

glucocorticoid treatment (D) and remain active. 

Regulation of the hematopoeitic niche by 

osteogenic hormones 

Stem cells are required for tissue homeostasis and re- 

generation. During ageing an exhaustion of stem cell self- 

renewal and decrease of the capacity to maintain tissue 

homeostasis occurs and accelerate the ageing process and 

the onset of age related diseases. We investigate the inter- 

action of hematopoeitic stem cells (HSCs) with their he- 

matopoeitic stem cell niche, comprised of either osteo- 

blasts or stromal/endothelial cells. We could show that 

estrogen treatment also enhances the numbers of hemat- 

opoeitic stem cells. Surprisingly, estrogen treatment only 

increases hematopoeitic stem cell fraction that is not as- 

sociated to bone, indicating that estrogens might act on 

the vascular niche. Currently we are testing the require- 

ment of the estrogen receptor alpha and beta for this ef- 

fect and if these receptors are acting cell autonomously in 

the hematopoeitic stem cells or in the cellular compart- 

ments of the niche. 

Metabolism and adipocyte differentiation 

Hormonal control is a major aspect of both tissue 

homeostasis and metabolism. Adipositas and insulin re- 

sistance are controlled by peripheral hormones. The cata- 

bolic actions of GCs contribute to these metabolic side ef- 

fects. Fat tissue in the obese individuals often secretes 

elevated amounts of GCs. To unravel these complex phe- 

nomena we investigate the effects of the GR on obesity 

and insulin resistance in mice carrying mutations of the 

A B 

C D 

Fig. 4: Bone formation determined by fluorescent calcein 

incorporation (A) is suppressed by glucocorticoids leading to 

osteoporosis (B). In mice lacking the glucocorticoid receptor 

in bone forming osteoblasts (C) the glucocorticoid 

suppression of bone formation is abrogated (D). 

GR in various cell types. The mechanisms involved in the 

metabolic syndrome are studied in in-house collabora- 

tions with the laboratories of H. Heuer, C. Calkhoven and 

F. Weih (“Metabolic Club”). The immunological aspects of 

our work which employ differentiation of blood cells in- 

flammatory models and the interactions of NF-kB with 

steroid receptors in several cell types are performed in 

close collaboration with the Weih lab. Inflammation- 

associated cancer and molecular aspects of GR-AP-1 inter- 

actions are analysed together with the Herrlich labora- 

tory. RNAi-screens to identify novel effectors of nuclear 

receptors are performed together with the Ploubidou, 

Morrison, Herrlich and Kaether laboratories. 

Author: Jan Tuckermann 

Phone: 0049-3641-656134 

E-mail: jan@fli-leibniz.de 

Lab members: Anita Neumann, Susanne Ostermay, Kerstin Jungert, 

Anna Kleyman, Anett Illing, Katrin Buder, Jan Tuckermann. 

Not pictured: Ulrike Baschant, Alexander Rauch, Sabine Hübner

Tissue section of a 

mouse cerebellum 

Out of Balance – 

Many specific molecules involved in DNA damage re- 

sponse play a critical role in DNA repair, cell-cycle control 

and the operation of apoptosis. Study of the mechanism 

of DNA damage response is important for improving our 

understanding of fundamental cellular processes and age- 

ing-related pathologies. Due to the restricted material for 

these processes in human studies, the experimental mod- 

els to mimicking human pathological symptoms is consid- 

ered highly desirable by the scientific community. Geneti- 

cally engineered animals, e.g. transgenic and knockout 

mice produced by gene-targeting technology in which one 

or more genes are modified, provide a powerful tool for 

scientists and clinicians in elucidating pathological 

processes in humans. These animal models also enhance 

our ability to evaluate a range of biomarkers prior to their 

clinical use and to validate environmental factors and 

therapeutic strategies in the treatment of pathological 

changes. 

Research activities 

Wang Lab 

How Genomic Instability Promotes Diseases and Ageing 

The hereditary material, DNA, is constantly being remodelled in our cells, e.g. in order to allow 

for production of active proteins or to repair damages of the DNA structure. Such processes are 

tightly controlled since they may lead to alterations of the DNA sequence containing the genetic 

information, which may in turn favour the cells transition to ageing, diseases and cancer. The labora- 

tory of Zhao-Qi Wang investigates the delicate balance of DNA damage responses that enable DNA 

repair processes while maintaining genomic stability and cellular homeostasis. 

Our laboratory generates genetically engineered mice 

and cells that serve as model systems for corresponding 

human conditions. In particular, we focus our studies on 

analysing: (I) the role of DNA damage response molecules 

[ATM, ATR, Fanconi anaemia proteins, MRE11/RAD50/ 

NBS1] in DNA repair pathways, genomic instability, tu- 

mourigenesis and tissue degeneration; (II) the biological 

function of poly(ADP-ribosyl)ation homeostasis modu- 

lated by PARP-1 and PARG in genomic stability, tissue in- 

jury and age-related pathologies. These two major topics, 

which are embedded in the overall interest in DNA repair 

shared by several labs at FLI, are outlined in the following. 

77

78 Wang Lab 

Blastocysts formed three days after fertilization of mouse 

oocytes 

DNA damage response and pathogenesis 

The Nijmegen breakage syndrome (NBS) is a rare in- 

herited syndrome characterised by DNA repair deficien- 

cies, chromosomal instability, microcephaly, mental retar- 

dation, immunodeficiency and cancer susceptibility. The 

product of the NBS gene (NBS1) forms a complex with 

RAD50 and MRE11 (M/R/N). This complex plays a multi- 

functional role in DNA damage signalling, ge- 

nomic stability and cell-cycle regulation. To in- 

vestigate the function of NBS1 in vivo and 

study the causal role of NBS1 mutations 

in the human NBS phenotype, we dis- 

rupted the Nbs1 gene in mice. Nbs1 ho- 

mozygous mutant mice die embryonically and 

Nbs1 heterozygous mutant mice developed various 

tumours in later life, suggesting that Nbs1 is an essential 

gene and plays a role in suppressing tumourigenesis. 

To further investigate the biological function of Nbs1 

and overcome embryonic fatality in Nbs1-null mice, we 

have generated “conditional” Nbs1 mutant mice using 

the Cre-loxP technique, which makes it possible to intro- 

duce a null mutation in specific tissues and cell types. 

When Nbs1 was deleted in neural tissues, the mice dis- 

played a combination of the neurological anomalies of 

NBS, ataxia telangiectasia (AT) and AT-like disorder (AT- 

LD), including microcephaly, growth retardation, cerebel- 

lar defects and ataxia due to proliferation and apoptosis 

defects. Nbs1-deficient neuroprogenitor cells contained 

more chromosomal breaks. Depletion of p53 significantly 

offset the neurological defects of these mice. These re- 

sults identify an essential role for NBS1 and the DNA dam- 

age response in neurological anomalies of NBS. Moreover, 

inactivation of the Nbs1 gene in the mouse lens causes 

progressive cataract formation due to disruption of epi- 

thelial cells differentiation in the lens. These studies dem- 

onstrate that defects in DNA damage response 

pathways play a causative role in the pathogene- 

sis of ageing phenotypes, including neurode- 

generation and progressive cataractogenesis. 

Finally, by constructing mouse Nbs1-null embry- 

onic stem cells and embryonic fibroblast cells, 

we found that Nbs1 determines the branching 

pathways of DNA repair by promoting homologous 

repair while repressing non-homologous end-joining. 

Our research is expected to yield key information on 

the mechanisms responsible for genomic instability, tel- 

omere stability, neuronal degeneration and immunodefi- 

ciency in genomic instability syndromes, namely A-T, A- 

TLD and NBS. Such knowledge has not been available so 

far and may facilitate the development of novel strategies 

for dealing with neurodegenerative components and ma- 

lignancy. 

Using a microinjection pipette (right), embryonic stem 

cells are injected into a blastocyst, which is held by a 

holding pipette (left). 

Right: Tissue section of a mouse cerebellum: We 

characterised defined genetic changes in the neurons 

that cause disturbed neurological function.

The homeostasis of poly(ADP-ribosyl)ation 

in genomic stability, tumourigenesis and 

tissue injury 

Poly(ADP-ribosyl)ation is an immediate cellular re- 

sponse to DNA damage. This post-translational modifica- 

tion is an extensive but transient modification modulated 

by poly(ADP-ribose) polymerase (PARP-1) and metabolized 

by poly(ADP-ribose) glycohydrolase (PARG). Genetic and 

molecular studies have demonstrated the involvement of 

PARP-1 in DNA repair, apoptosis, genomic stability and 

proliferation, as well as in transcription regulation. In col- 

laboration with others, we have found that in animal 

models, PARP-1 deficiency promotes tumourigenesis of 

the liver, breast and brain and accelerates ageing, but pro- 

tects mice from diabetes, strokes and inflammations. A 

single nucleotide polymorphism in humans is associated 

with breast cancer. Although PARP-1 is dispensable in the 

repair of DNA double-strand breaks, it is involved in the 

repair and reactivation of stalled replication forks. 

To further study the homeostasis of poly(ADP)ribo- 

syl)ation, we have generated mice lacking the principal 

isoform (110 kD) of PARG (PARG110) and found that these 

mutant mice are hypersensitive to alkylating agents and 

ionizing radiation. These mice however were also suscep- 

tible to diabetes, stroke and endotoxic shock. PARG110 

knockout mice are protected from renal injury, intestinal 

ischemia-reperfusion damage, experimental spinal-cord 

trauma and DNBS-induced colon injury, apparently due to 

Lab members: Tangliang Li, Christopher Bruhn, Ralph Gruber, 

Zhao-Qi Wang, Tjard Joerß, Mareen Welzel, Bénazir Siddek, 

Kristin Kiesow, Laura Perucho Aznar, Wookee Min, Amal Saidi, 

Sandra Orthaus, Anja Krüger. Not pictured: Mikhail Sukchev 

a function of PARG in inflammation response. In addition, 

cellular studies showed delayed DNA repair, concomitant 

with increased sister chromatid exchange, micronuclei 

and chromosomal aberrations, as well as hyper-amplifica- 

tion of centrosomes, all of which are hallmarks of ge- 

nomic instability. These results suggest that modulation 

of poly(ADP-ribosyl)ation is important in genomic stabil- 

ity, inflammatory response, tissue homeostasis, and the 

prevention of carcinogenesis. These studies also suggest 

potential use of PARG inhibitors in therapy. 

Author: Zhao-Qi Wang 

Phone: 0049-3641-656415 

E-mail: zqwang@fli-leibniz.de 

79

80 Weih Lab 

Vital Communication: The NF-κB Signal Transduction 

Pathway in the Immune System 

An intact and functional immune system is essential for maintaining a good health status in 

particular in the elderly. In the immune system‘s response to pathogenic agents, the activation 

of specific genes by transcription factors plays a crucial role that is significant for development and 

functioning of the immune system itself. This is also true for inflammatory processes and cancer 

genesis. The lab of Falk Weih studies how one very important family of transcription factors 

influences the immune system and indicates how knowledge of this kind can help us to better 

understand age-related immune deficiencies and disease. 

Transcription factors are proteins with a positive or 

negative effect on the expression of genes. Certain tran- 

scription factors – those of the Rel/NF-κB family – play an 

important role in immune responses, inflammatory pro- 

cesses, the regulation of apoptosis and cancer. Using 

genetically altered mice, we are analysing NF-κB signalling 

in both normal development and pathological alterations 

of the immune system. Our work centres on the activation 

of the NF-κB family member “RelB” via the recently 

described “alternative” pathway. 

The development of important organs in 

the immune system 

Spleen, lymph nodes and Peyer’s patches in the small 

intestine are indispensable for an effective immune re- 

sponse to pathogenic agents invading the body. They rep- 

resent strategic locations where native T and B lym- 

phocytes are activated by so-called antigen presenting 

Thymus 

Appendix 

cells. The analysis of mice that lack the RelB protein 

shows that RelB controls the development of the micro- 

structure of the spleen. In the absence of RelB, lymph 

nodes remain in a rudimentary state, while Peyer’s 

patches in the small intestine do not develop at all. Bio- 

chemical and genetic studies have demonstrated that 

RelB has to be activated by the lymphotoxin-ß receptor 

(LTßR) for secondary lymphoid organs like the spleen, 

lymph nodes and Peyer’s patches to develop normally. In 

addition to “alternative” RelB activation there is also acti- 

vation of RelA via the “classical” signalling pathway (see 

diagram on next page). This pathway can also be acti- 

vated by the LTßR and loss of RelA also impairs normal de- 

velopment of secondary lymphoid organs. At present we 

are trying to understand the specific roles of RelA and 

RelB in the development of lymphoid organs and in au- 

toimmunity. In addition, we are looking for target genes 

that are transcriptionally regulated by RelA or RelB down- 

stream of the LTßR. 

Palatine Tonsils 

Tonsils 

Lymph Nodes 

Lymphatics 

Spleen 

Peyer“s 

Patches 

Bone Marrow

p100 

α α 

p52 

p52 

P P 

RelB 

RelB 

RelB 

LTβR 

Alternative NF-κB 

Activation 

NIK 

Pathway 

IKKα activation 

p100 

phosphorylation 

p100 

ubiquitination 

and processing 

p100 

C-term. 

Lymphoid organ development 

Adaptive immunity 

Regulation of natural killer T cell and B 

cell development by NF-κB 

Ub Ub 

P P 

Natural killer T cells (NKT cells) are a small but impor- 

tant subpopulation of T cells in the immune system. 

Along with an invariant T cell receptor, NKT cells also carry 

the NK1.1 marker. Once activated, they produce certain 

messenger substances of the immune system, the cy- 

tokines IL-4 and IFN-γ, and it has been proposed that they 

repress autoimmune disorders and mount anti-tumour re- 

sponses. In experiments with mice we have been able to 

show that the activation of the RelB protein in stromal 

cells of the thymus – the natural training ground for all T 

cells – is indispensable for natural killer T cells to develop 

normally. The classical NF-κB signalling pathway, for its 

part, controls the development of precursor cells into ma- 

LTα 1 β 2 

Cytoplasm 

UbUb Ub 

P P 

Ub 

Bα 

Iκ 

Nucleus 

MEKK3 

γ 

α β 

P P 

Classical 

IκBα 

p50 RelA 

p50 RelA 

p50 RelA 

IKKβ activation 

IκBα 

phosphorylation 

IκBα 

ubiquitination 

and proteasomal 

degradation 

Cell survival, inflammation 

Innate immunity 

ture NKT cells in a cell-autonomous way. At present we 

are investigating the molecular pathways through which 

RelA regulates the development of NKT cells in the thy- 

mus. 

Based on complex signalling pathways, organs and 

specialised cells of the immune system are generated 

during embryogenesis. 

We are also studying how development of B cells in 

the bone marrow and the differentiation of various B cell 

subpopulations in the spleen take place. Our results indi- 

cate that the constitutive activation of the alternative NF- 

κB pathway and RelB activity interferes with I) early B cell 

development in the bone marrow; II) normal differentia- 

tion of B cell subpopulations in the spleen; and III) a well 

structured and functional splenic marginal zone. Thus, 

81

82 Weih Lab 

alternative NF-κB activation has to be tightly controlled to 

prevent aberrant B cell development and a disorganised 

spleen microarchitecture. 

Atherosclerosis and neurodegenerative 

diseases 

Atherosclerosis constitutes the single most important 

contributor to cardiovascular disease, the leading cause of 

death and illness in developed countries. In collaboration 

with the Institute of Vascular Medicine at the Friedrich- 

Schiller-University Jena we have begun to investigate the 

role of lymphotoxin signalling for the development of so- 

called tertiary lymphoid organs in the aorta adventitia 

and for atherosclerosis in aged hyperlipidemic mice. 

We also use mouse models to investigate cerebral 

ischemia (stroke), another leading cause of death and dis- 

ability worldwide. In collaboration with the Institute of 

Pharmacology in Heidelberg we started to investigate the 

role of the NF-κB family members RelA and RelB, repre- 

senting the classical and alternative NF-κB activation 

pathway, respectively, in induced focal cerebral ischemia. 

We also collaborate with a neurology lab in Jena on the 

role of NF-κB in neuroprotection and regeneration of the 

injured optic nerve. 

The Peyer’s patches of the small intestine are structures of the immune system located in a strategically 

important place: Invading pathogens here encounter defending immune cells. Peyer’s Patches cannot develop 

without the protein RelB: The images show Peyer’s Patches of healthy mice (wild-type) and of mice whose gene 

for this important protein has been switched off (relB-/-). 

In-house collaborations include work on the role of 

the Nijmegen Breakage Syndrome (Nbs-1) gene in the im- 

mune system (Wang laboratory) as well as NF-κB func- 

tion in osteoblasts and in septic shock models (Tucker- 

mann laboratory). 

Author: Falk Weih 

Phone: 0049-3641-656048 

E-mail: fweih@fli-leibniz.de 

Lab members: Falk Weih, Debra Weih, Iwona Powolny, Verena Wolf, 

Elke Meier, Feng Guo, Simone Tänzer, Marc Riemann, Agnes Lovas, 

Alexander Werner

Chairman: Wolfram Eberbach 

 

 


Head of Administration: Daniele Barthel 

 

 


 

H. Lekscha; G.Bergner; E.Stöckl 

 

Zhao-Qi Wang 

 


 


 

Peter Hemmerich 

 

Matthias Platzer 

 

Jan Tuckermann 

 

Falk Weih 

 

Heike Heuer 

 

 

K.H. Gührs B. Schlott 

 

Matthias Görlach 

 


 

Christian Hoischen 

 

C. Calkhoven, H. Heuer, C. Kaether 

 

M. Than 

 

Benita Rost 

as of January 2008 

 

Chairman: Piet Borst 

 

 

 

 

Diana Kirchhof 

 

Swen Löhle 

 

 

 

Picture credits 

Organisation Chart 

Pictures and graphics were supplied by the authors 

and members of FLI, unless stated otherwise. 

Portraits were taken by Gerhard Müller, FLI, and 

Rolf Hühne, FLI. 

Peter Scheere (FSU) contributed the portraits on 

pages: 31, 33, 36, 39, 40, 42, 43, 45, 46, 48, 54, 55, 57, 

60, 63, 68, 71, 74, 76, 77, 79, 80, 82. 

The editor FLI intended to obtain permission for 

using copyrighted material wherever possible. 

We thank the following contributors: 

Front cover: Herbert Thum 

Page 4: Birgitta Kowsky 

Page 5, portrait gallery: Herbert Thum 

Page 6: We thank Mr. S. Lipmann, son of 

Fritz Lipmann, for releasing the material 

Paage 7, demography chart: J. Vaupel, K.G. von 

Kistowski (MPI Rostock) 

Page 12: Tino Zippel, Osttühringische Zeitung 

Page 14, portrait: Eckhardt Hoenig 

Pages 15 and 16, Dictyostelium: M. J. Grimson & 

R. L. Blanton 

Page 19, graphics: Herbert Thum 

Page 62: Andreas Bolzer, Gregor Kreth, Daniela 

Koehler, Kaan Saracoglu, Christine Fauth, Stefan 

Müller, Roland Eils, Christoph Cremer, Michael 

Speicher, Thomas Cremer 

Page 70: We thank the journal „RNA“ 

(www.rnajournal.org) for release of cover pictures 

Page 80, graphics: Herbert Thum 

Back cover: Herbert Thum 

83

Imprint 

Publisher 

Leibniz Institute for Age Research – 

Fritz Lipmann Institute (FLI) 

Beutenbergstraße 11 

07745 Jena, Germany 

www.fli-leibniz.de 

Concept and Realisation 


Scientific Coordinator 

Text Editing and Translations 

Andrew Jenkins 

Debra Weih 

Claudia Eberhard-Metzger 

www.eberhard-metzger.de 

Layout and Design 

Herbert Thum 

Viskon Kommunikation & Design 

www.viskon.de 

Print 

Nino Druck GmbH 

Neustadt/Weinstraße, Germany

The Puzzle of Ageing - Leibniz Institute for Age Research

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