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JCB THE JOURNAL OF CELL BIOLOGY 2012 HIGHLIGHTS www.jcb.org

JCB

THE JOURNAL OF CELL BIOLOGY

2012

HIGHLIGHTS

www.jcb.org

JCB

THE JOURNAL OF CELL BIOLOGY

EDITOR-IN-CHIEF

TOM MISTELI

EDITORS

PIER PAOLO DI FIORE

ELAINE FUCHS

ALAN HALL

REBECCA HEALD

IRA MELLMAN

JODI NUNNARI

LOUIS F. REICHARDT

KENNETH M. YAMADA

JUNYING YUAN

NEWS EDITOR

BENJAMIN SHORT

REVIEWS EDITOR

PRIYA PRAKASH BUDDE

EDITORIAL BOARD

JOHN AITCHISON

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BEN BARRES

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TONY BRETSCHER

MARIANNE BRONNER

ERIC J. BROWN

DON W. CLEVELAND

PASCALE COSSART

GAUDENZ DANUSER

PIETRO DE CAMILLI

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ABBY DERNBURG

ARSHAD DESAI

RAY DESHAIES

IVAN DIKIC

STEVE DOXSEY

WILLIAM EARNSHAW

SCOTT EMR

JEFFREY ESKO

HIRONORI FUNABIKI

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EXECUTIVE EDITOR

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email: lwilliams@rockefeller.edu

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SYNAPTIC SYSTEMS

www.sysy.com

THE WORLD OF

SYNAPTIC SYSTEMS

RESEARCH TOOLS

FOR NEUROSCIENCE

AND CELL BIOLOGY

Shining a Spotlight on the Best Cell Biology

Liz Williams 1 and Tom Misteli 2

1 Executive Editor and 2 Editor-in-Chief, The Journal of Cell Biology

The past year has been an exciting one at JCB

as we have continued to publish some of the

most important and infl uential papers across

cell biology. With the onslaught of information in all

of our lives today, however, the days of casually fl ipping

through a journal to see what is new outside of the

particular fi elds we work in is a rare event. We know

full well how hard it can be to keep up. But there is

so much good science out there! Where should today’s

busy scientist fi nd inspiration for the next phase of his

or her research? Science so often is driven by serendipity

and happenstance—with inspir ation often coming from

that quick conversation over lunch at a conference,

that tiny nugget of information buried in the paper

chosen for journal club, or a question asked at the

seminar you almost didn’t fi nd time to attend.

Welcome

The JCB Special Issue, which we distribute each year at the American Society for Cell Biology Annual Meeting, continues

to be a favored resource for a quick “catch up” on some of what has been happening in the world of cell biology over the previous

six months. With this JCB Highlights Issue we are continuing a tradition of providing a mid-year “catch up”. This Issue shines a

spotlight on some of the best original research and review content from the past six months in JCB. We encourage you to take a look,

pass this issue around, and then visit www.jcb.org to explore these stories and more in greater depth. While there, also check out our

highly popular multimedia teaching tools: biobytes audiocasts, biosights videocasts, biowrites blog, and our Journal Club packs. We

hope the work you see here provides a useful portal to a wealth of new ideas for your own work.

See JCB Highlights 2012 online at www.jcb.org/site/highlights2012 and

access the FREE full text of all highlighted articles at www.jcb.org.

JCB Highlights 2012

By Michael R. Green, Howard Hughes Medical Institute, University

of Massachusetts Medical School and Joseph Sambrook,

Peter MacCallum Cancer Institute, Melbourne, Australia

Molecular Cloning: A Laboratory Manual has always been the

one indispensable molecular biology laboratory manual

for protocols and techniques. The fourth edition of this classic

manual preserves the detail and clarity of previous editions

as well as the theoretical and historical underpinnings of

the techniques presented. Ten original core chapters reflect

developments and innovation in standard techniques and introduce

new cutting-edge protocols. Twelve entirely new chapters

are devoted to the most exciting current research strategies,

including epigenetic analysis, RNA interference, genome

sequencing, and bioinformatics. This manual is essential for both

the inexperienced and the advanced user.

2012, 1,890 pp., illus., appendices, index

Cloth (three-volume set)

$475 ISBN 978-1-936113-41-5

Paperback (three-volume set)

$365 ISBN 978-1-936113-42-2

Contents

VOLUME 1

Part 1 Essentials

1. Isolation and Quantification

of DNA

2. Analysis of DNA

3. Cloning and Transformation

with Plasmid Vectors

4. Gateway Recombinational

Cloning

5. Working with Bacterial Artificial

Chromosomes and Other

High-Capacity Vectors

6. Extraction, Purification,

and Analysis of RNA from

Eukaryotic Cells

7. Polymerase Chain Reaction

8. Bioinformatics

VOLUME 2

Part 2 Analysis and Manipulation of

DNA and RNA

9. Quantification of DNA and

RNA by Real-Time Polymerase

Chain Reaction

10. Nucleic Acid Platform

Technologies

11. DNA Sequencing

12. Analysis of DNA Methylation

in Mammalian Cells

13. Preparation of Labeled DNA, RNA,

and Oligonucleotide Probes

14. Methods for In Vitro Mutagenesis

Part 3 Introducing Genes into Cells

15. Introducing Genes into Cultured

Mammalian Cells

16. Introducing Genes into

Mammalian Cells: Viral Vectors

VOLUME 3

Part 4 Gene Expression

17. Analysis of Gene Regulation Using

Reporter Systems

18. RNA Interference and Small

RNA Analysis

19. Expressing Cloned Genes for

Protein Production, Purification,

and Analysis

Part 5 Interaction Analysis

20. Cross-Linking Technologies for

Analysis of Chromatin Structure

and Function

21. Mapping of In Vivo RNA-Binding

Sites by UV-Cross-Linking

Immunoprecipitation (CLIP)

22. Gateway-Compatible Yeast One-

Hybrid and Two-Hybrid Assays

Appendices

1. Reagents and Buffers

2. Commonly Used Techniques

3. Detection Systems

4. General Safety and Hazardous

Material

Index

SELECTED HIGHLIGHTS

10 • Licensing factors lose their credentials

Mitch Leslie

• Cell death helps give closure

Ben Short

• Meet the neighbors

Ben Short

11 • VPS35 leaves endosomes lost in transition

Mitch Leslie

• Lipid droplets fatten up with Fsp27

Ben Short

• The actomyosin ring bulks up

Mitch Leslie

12 • Stress fi bers guide focal adhesions to maturity

Ben Short

13 • Aurora B is no Ska fan

Mitch Leslie

• Wait, save that integrin!

Mitch Leslie

• Slow but steady for NF- � B

Mitch Leslie

14 • BRCA1 touches up microRNAs

Mitch Leslie

• No crystal structure? No problem

Mitch Leslie

• CUPS provide a handle on Acb1 secretion

Ben Short

15 • Big enough for two

Ben Short

16 • References for selected highlights

REVIEWS

17 The extracellular matrix: A dynamic niche in cancer progression

Pengfei Lu, Valerie M. Weaver, and Zena Werb

J. Cell Biol. 2012. 196:395–406.

29 Tracing epithelial stem cells during development, homeostasis,

and repair

Alexandra Van Keymeulen and Cédric Blanpain

J. Cell Biol. 2012. 197:575–584.

JCB

THE JOURNAL OF CELL BIOLOGY

HIGHLIGHTS 2012

Cover design by Liz Williams.

Images (clockwise from top right):

courtesy of Yvonne Beckham and Patrick

Oakes; courtesy of Parveen Suraneni;

courtesy of Anjali Sarkar; courtesy of

Stephan Huveneers; courtesy of Jacob

Rullo and Henry Hong.

A GE Healthcare Company

Real speed

Real time 3D imaging

Real live cell super-resolution

Really

DeltaVision OMX ®

with the Blaze SIM Module

Experience a new dimension

in super-resolution imaging

Learn more at www.super-resolution.com

Macrophage (RAW) cell membranes stained with WGA Alexa488 and 100 nm

glass beads (red) that have been endocytosed by the cell - movie courtesy of

Lynne Turnbull, Eileen McGowan and Cynthia Whitchurch, Microbial Imaging

Facility, University of Technology, Sydney.

EXECUTIVE DIRECTOR

MIKE ROSSNER

FINANCE DIRECTOR

RAYMOND T. FASTIGGI

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THE JOURNAL OF CELL BIOLOGY (ISSN 0021-9525) IS PUBLISHED BIWEEKLY

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POSTMASTER:

SEND ADDRESS CHANGES TO THE JOURNAL OF CELL BIOLOGY, CIRCULATION

DEPARTMENT, THE ROCKEFELLER UNIVERSITY PRESS, 1114 FIRST AVENUE,

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��

��

� ��

��

��

Announcing Keystone Symposia

2012–2013 Conferences

Pulmonary Vascular Disease and Right Ventricular Dysfunction:

Current Concepts and Future Therapies

��

Aging and Diseases of Aging

��

Immunological Mechanisms of Vaccination

��

Type 2 Immunity: Initiation, Maintenance, Homeostasis and Pathology

joint with: Pathogenic Processes in Asthma and COPD

��

Multiple Sclerosis

��

New Frontiers in Cardiovascular Genetics beyond GWAS

��

Frontiers of NMR in Biology

��

Hematopoiesis

��

Emerging Topics in Immune System Plasticity:

Cellular Networks, Metabolic Control and Regeneration

��

Plant Abiotic Stress and Sustainable Agriculture:

Translating Basic Understanding to Food Production

��

Noncoding RNAs in Development and Cancer

��

Malaria

��

Metabolic Control of In�lammation and Immunity

��

Antibodies as Drugs joint with:

Cancer Immunology and Immunotherapy

��

Adipose Tissue Biology joint with:

Diabetes – New Insights into Mechanism of Disease and Its Treatment

��

Mitochondria, Metabolism and Myocardial Function –

Basic Advances to Translational Studies

��

Neurogenesis joint with:

New Frontiers in Neurodegenerative Disease Research

��

Lung Development, Cancer and Disease

��

The Gut Microbiome: The Effector/Regulatory Immune Network

��

B Cell Development and Function joint with: HIV Vaccines

��

Autophagy, In�lammation and Immunity

��

Scholarships are available to students and postdoctoral fellows. Abstract and

scholarship deadlines are four months before conferences begin, and early registration

deadlines, saving US$150 on later fees, precede conferences by two months.

Visit www.keystonesymposia.org/2013meetings for more information.

Nutrition, Epigenetics and Human Disease

��

Myeloid Cells: Regulation and In�lammation

��

Stem Cell Regulation in Homeostasis and Disease

��

PI 3-Kinase and Interplay with Other Signaling Pathways

joint with: Tumor Metabolism

��

Structural Analysis of Supramolecular Assemblies by Hybrid Methods

��

Understanding Dendritic Cell Biology to Advance Disease Therapies

��

DNA Replication and Recombination

joint with: Genomic Instability and DNA Repair

��

Growing to Extremes: Cell Biology and Pathology of Axons

��

Host Response in Tuberculosis joint with:

Tuberculosis: Understanding the Enemy

��

Precision Genome Engineering and Synthetic Biology:

Designing Genomes and Pathways

��

Neuronal Control of Appetite, Metabolism and Weight

��

RNA Silencing

��

Epigenetic Marks and Cancer Drugs

��

Molecular Clockworks and the Regulation

of Cardio-Metabolic Function

��

Immune Activation in HIV Infection:

Basic Mechanisms and Clinical Implications

��

Nuclear Receptors and Friends:

Roles in Energy Homeostasis and Metabolic Dysfunction

��

Immunopathology of Type 1 Diabetes joint with:

Advances in the Knowledge and Treatment of Autoimmunity

��

Cardiac Remodeling, Signaling, Matrix and Heart Function

��

Plant Immunity: Pathways and Translation

��

Positive Strand RNA Viruses

��

The Innate Immune Response in the Pathogenesis

of Infectious Disease

��

The Hippo Tumor Suppressor Network:

From Organ Size Control to Stem Cells and Cancer

��

Human Genomics and Personalized Medicine

��

Selected Highlights

10.1083/jcb.1962iti1jcb.1962iti1Mitch Lesliemitchleslie@comcast.netjcb.1962iti1fig1.epsIn

This IssueNewsLicensing factors

lose their credentials

Sonneville et al.

reveal how C.

elegans embryos

control replication li censing

factors to pro mote

As anaphase progresses (left to right), effi cient DNA duplication

more and more Mcm3 protein (glowing while pre venting the

dots) attaches to the DNA.

genome from being copied

more than once.

A cell starts preparing to duplicate its DNA before it has

fi nished dividing. Beginning in anaphase, the cell fl ags replication

origins where DNA replication will commence. Licensing factors

cooperate to mark replication origins by clamping the Mcm2–7

complex around the DNA. Once a cell has tagged a large number

of replication origins, it shuts down licensing before S phase so

that each section of DNA will be duplicated only once.

10.1083/jcb.1956iti3jcb.1956iti3Ben Shortbshort@rockefeller.edujcb.1956iti3fi g1.epsIn This IssueNewsCell death

helps give closure

By visualizing the dynamics of apoptosis in living mouse

embryos, Yamaguchi et al. reveal that cell death helps

drive the morphogenetic movements required for neural

tube closure (NTC).

NTC is a vital early step in the development of the central

nervous system. A fl at group of cells called the neural plate bends

in the middle so that the sides of the plate curve around to meet

and fuse with each other, forming the neural tube. Many mouse

embryos lacking key apoptosis proteins, such as Apaf-1 or

Caspase-3, show defects in NTC in their cranial region, but how

cell death contributes to this process is unclear.

Yamaguchi et al. generated transgenic mice expressing

a FRET-based apoptotic reporter whose signal is decreased when

activated caspases cleave the link between two fluorescent

proteins. Using this reporter, the researchers identifi ed two types

of apoptotic cells in embryonic brains undergoing NTC. Some cells

died and fragmented rapidly, whereas others—particularly near

the tips of the folding neural plate—persisted for longer without

breaking apart.

Both types of apoptotic cells were absent from mouse embryos

lacking Apaf-1 and Caspase-3. In these animals, neural plate bending

was reduced, thereby delaying NTC. Senior authors Yoshifumi

Yamaguchi and Masayuki Miura think that the death, and subsequent

extrusion, of cells from the tips of the neural plate may generate forces

that help to shape the developing tissue and facilitate cranial NTC.

Alternatively, the two types

of apoptotic cells may direct

this developmental process

by sending different signals

to their surviving neighbors.

Ben Short

Several apoptotic cells (blue) persist

at the edge of the neural plate as it

folds to form the neural tube.

Yamaguchi, Y., et al. 2011. J. Cell Biol.

http://dx.doi.org/10.1083/jcb.201104057.

Using live-cell imaging, Sonneville et al. followed the

dynamics of licensing factors in C. elegans. The researchers

propose that the specific behavior of the proteins makes

licensing more effi cient. Two of the main licensing factors, the

origin recognition complex (ORC) and CDC-6, attach to DNA

independently, potentially speeding up replication licensing.

And the binding of the Mcm2–7 proteins to DNA encouraged

the release of CDC-6 and the ORC, allowing them to move on

and mark other origins.

Sonneville et al. also determined how a cell curtails replication

licensing to stop double duplication of its DNA: the cell

expels CDC-6 and ORC from the nucleus during interphase, a

process that required the nuclear export factor XPO-1. Depleting

this molecule slowed removal of CDC-6 and ORC components

from the nucleus and allowed DNA rereplication. Mitch Leslie

Sonneville, R., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201110080.

10.1083/jcb.1966iti3jcb.1966iti3Ben Shortbshort@rockefeller.edujcb.1966iti3fi g1.epsIn This IssueNewsMeet the

neighbors

Roux et al. report a

new way to screen for

protein–protein interactions

in mammalian cells.

Existing methods for

probing protein inter actions

have their limitations. A yeast

two-hybrid screen, for example,

involves ex pressing

proteins in a non-native cell

type that may not fold or

A BirA–lamin-A fusion protein (red)

attaches biotin (green) to nearby

components of the nuclear envelope.

modify them correctly. Biochemical “pull-down” approaches, on

the other hand, are limited by protein solubility and can miss

weak or transient interactions. Roux et al. developed a new

method called proximity-dependent biotin identifi cation, or BioID,

which relies on a promiscuous mutant of the bacterial biotin

ligase BirA that biotinylates nearby primary amines, such as lysine

residues in neighboring proteins.

The researchers fused the mutant ligase to human lamin-A, an

insoluble component of the protein meshwork that underlies the inner

nuclear membrane. When the fusion protein was expressed in cells,

it localized to the nuclear envelope and biotinylated nearby proteins,

which could be purifi ed on biotin-binding beads and identifi ed by

mass spectrometry. This approach detected many nuclear membrane

proteins and nuclear pore complex components known to associate

with lamin-A. It also identifi ed a previously uncharacterized protein

that the authors localized to the nuclear envelope and named soluble

lamina-associated protein of 75 kD, or SLAP75.

BioID identifi es both a protein’s binding partners and its near

neighbors, says senior author Kyle Roux. In addition to using the

technique with different target proteins, Roux wants to examine

how disease-linked mutations in lamin-A alter the protein’s

association profi le. Ben Short

Roux, K.J., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201112098.

10.1083/jcb.1955iti1jcb.1955iti1Mitch Lesliemitchleslie@comcast.netjcb.1955iti1fig1.epsIn

This IssueNewsVPS35 leaves

10.1083/jcb.1956iti1jcb.1956iti1Ben Shortbshort@rockefeller.edujcb.1956iti1fi g1.epsIn This IssueNewsLipid droplets

endosomes lost in transition

Relative to control cells (left), cells short

on VPS35 (right) accumulate more

BACE1 (red) in endosomes (green).

Sluggish recycling

of a protein-slicing

enzyme could promote

Alzheimer’s disease

(AD), Wen et

al. show.

A�, the protein that

accumulates in the brains

of AD patients, is formed

when enzymes cut up its

parental protein, known as amyloid precursor protein. One of

those enzymes is �-secretase or BACE1. BACE1 cycles between

the Golgi apparatus and the plasma membrane, traveling through

endosomes on the way. A protein complex called the retromer

helps transport proteins from endosomes to the Golgi. Previous

studies have found reduced levels of two retromer components,

including the protein VPS35, in the brains of AD patients.

fatten up with Fsp27

An adipocyte protein promotes the growth of lipid droplets

(LDs) by facilitating lipid transfer from smaller to

larger droplets, Gong et al. report.

Consisting of a neutral lipid core surrounded by a monolayer

of phospholipids and associated proteins, LDs serve as the cell’s fat

storage depots, particularly in adipocytes where they grow to extra

large sizes. How LDs grow is unknown, but adipocytes lacking the

LD-associated protein Fsp27 have many small droplets instead of a

single large one.

Gong et al. found that Fsp27 concentrated at the contacts

between LDs and that this localization depended on the protein’s

C-terminal domain. Photobleaching experiments revealed that

LDs in contact with each other traded neutral lipids but that this

exchange was abolished in adipocytes lacking Fsp27. Moreover,

the researchers identifi ed three lysine residues—which may form

part of an amphipathic helix in Fsp27’s C terminus—that were

required for lipid exchange and LD growth.

10.1083/jcb.1955iti2jcb.1955iti2Mitch Lesliemitchleslie@comcast.netjcb.1955iti2fig1.epsIn

This IssueNewsThe actomyosin

ring bulks up

Calvert et al. reveal

that large fungal

cells have more

myosin II associated with

their contractile rings

Purple in this heat map indicates that than do smaller cells,

there is more myosin II in a larger which allows them to

contractile ring (left) than in a smaller constrict the ring faster

one (right).

during cytokinesis.

A recent study of C. elegans embryos showed that the time

required for the contractile ring to close during cytokinesis was

about the same no matter the size of the cell, which means the

ring must tighten faster in larger cells. Scientists have been keen

to test whether girth affects constriction rate in other species. The

obstacle has been fi nding organisms whose dividing cells differ

enough in size.

To fi nd out whether VPS35 affects AD, the team crossed

two mouse lines to create animals that are prone to many AD

symptoms and generate half the normal amount of VPS35. The

mice displayed AD-like abnormalities earlier than their parental

strains, and their brains accumulated more A�.

Cells lacking VPS35 carried extra BACE1 in their endosomes.

BACE1 is more active in the acidic interior of endosomes

than in the more basic surroundings of the Golgi apparatus.

Thus, by leaving more BACE1 trapped in endosomes, the decline

in VPS35 levels could spur the formation of more A�.

Although no VPS35 mutations have so far turned up in AD

patients, the protein’s level in the brain dwindles with age

in mice. The researchers suspect that certain AD risk factors,

such as oxidative stress, also diminish VPS35 levels in the brain.

Mitch Leslie

Wen, L., et al. 2011. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201105109.

Although LDs exchanged lipids

bidirectionally, net lipid transfer

occurred from the smaller to the

larger droplet of an LD pair, leading

to shrinkage of the smaller droplet

and growth of the larger one. This directional

transfer is probably driven

by the higher internal pressure within

smaller droplets, which would force

lipids into the larger LD.

How Fsp27 facilitates this transfer

remains unclear, but senior author

Peng Li thinks that the protein may

Fsp27 (red) localizes to the

site of contact (arrowhead)

between lipid droplets (green)

in adipocytes.

help to connect LDs and form a pore to allow lipid movement. The

next question, she says, is to identify additional proteins that work

with Fsp27 to boost LD growth. Ben Short

Gong, J., et al. 2011. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201104142.

JCB Highlights 2012 11

Calvert et al. recognized a candidate for such a study—

the fungus Neurospora crassa. Its filaments, or hyphae, can

vary up to fourfold in diameter and undergo a process called

septation that’s somewhat akin to mitosis.

As in nematodes, the ring closed faster in larger hyphae.

But N. crassa differed from nematodes in a couple of ways.

In N. crassa, the contractile rings of larger cells started out with

more myosin II than the rings of smaller cells, which could

drive faster constriction. Indeed, reducing the amount of ringassociated

myosin II slowed the rate of constriction. In addition,

the amount of myosin II in the ring remained constant as the

ring tightened, instead of declining as in nematodes. Therefore,

in N. crassa, the design and mechanics of the contractile ring

vary with size. Future studies will investigate whether the same

relationship holds in other species. Mitch Leslie

Calvert, M.E.K., et al. 2011. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201101055.

Selected Highlights

10.1083/jcb.1963ifBen Shortbshort@rockefeller.eduIn FocusNewsStress fibers

guide focal adhesions to maturity

Study suggests that actin bundles serve as templates for adhesion growth.

Integrin-based focal adhesions initially

form at the leading edge of a

migrating cell and then mature into

larger structures that stably attach and

transmit force to the extracellular matrix

(ECM). Oakes et al. describe how this

maturation process is guided by the actin

stress fi bers that assemble at nascent

adhesions (1).

jcb.1963iffi g1.epsMaturing focal adhesions grow in size

and change their protein composition, and,

in fi broblasts, they eventually develop into

specialized fi brillar adhesions that can remodel

the ECM (2). Maturation depends

on the motor protein myosin II, which puts

the adhesions under tension and promotes

the assembly of adhesion-associated actin

bundles called radial stress fi bers. “It’s

been presumed that maturation is entirely

tension dependent and that the stress fi bers

are important for transmitting the myosingenerated

forces to the adhesions,” explains

Margaret Gardel, from the University

of Chicago. But myosin generates tension

and radial stress fi bers simultaneously,

making it diffi cult to dissect the roles of

these two processes in adhesion maturation.

“We wanted to investigate whether

the radial stress fi bers that form at focal

adhesions are important for force transmission

and the maturation

process,” Gardel recalls.

Gardel and colleagues,

led by Patrick Oakes and

Yvonne Beckham, blocked

the formation of adhesionassociated

stress fi bers by

inhibiting either the actinnucleating

formin Dia1 (3)

or the fi lament-bundling

protein �-actinin (4). Cells

treated with inhibitors or

shRNAs targeting these proteins failed to assemble

radial stress fi bers, despite having

normal levels of active myosin II. Other actin

structures in the cell were unaffected.

Cells lacking radial stress fi bers

formed small focal adhesions, which,

says Gardel, “were still under a lot of

tension, so the stress fi bers aren’t that

“Radial stress

fibers… serve

as structural

templates to

recruit other

focal adhesion

proteins.”

important for force transmission.” The

adhesions’ small size, however, suggested

that, in the absence of radial

stress fi bers, tension isn’t suffi cient to

drive adhesion maturation. Indeed, key

components of mature focal adhesions

weren’t recruited when stress fi ber formation

was inhibited, and fi broblasts

lacking Dia1 or �-actinin failed to assemble

fibrillar adhesions capable of

remodeling the ECM.

“So the recruitment of proteins [to

form mature adhesions] is strongly correlated

to the assembly of actin bundles at

the focal adhesion plaque,”

Gardel says. Adhesions fail

to mature in the absence of

radial stress fi bers, even

though myosin II continues

to exert signifi cant amounts

of force on nascent cell–

matrix attachments.

Oakes et al. then examined

the effect of myosin

II–dependent tension

on focal adhesion maturation.

The researchers limited myosin II

activity by treating cells with increasing

concentrations of a Rho kinase inhibitor.

Though some myosin II activity is required

to form mature adhesions, motor

function and intracellular tension could

be reduced by as much as 80% without

inhibiting adhesion growth and maturation.

FOCAL POINT

(Left to right) Jonathan Stricker, Yvonne Beckham, Margaret Gardel, and Patrick Oakes reveal

that focal adhesion–associated stress fi bers aren’t required to transmit force from the actin

cytoskeleton to the extracellular matrix but they are essential for the growth and maturation

of adhesions into stable cell–matrix attachments. The authors suggest that the stress fi bers

form a structural template that helps recruit focal adhesion components. Heat maps show that

autophosphorylated focal adhesion kinase is more enriched at focal adhesions in a control cell

(center) than in a cell lacking radial stress fi bers (right).

“So you only need a really minimal

threshold of tension,” Gardel says.

Gardel and colleagues think that this

minimal level of myosin II activity is required

to drive a “retrograde fl ow” of actin

fi laments away from the cell’s leading

edge. These fi laments are captured at nascent

cell adhesions and converted by

Dia1 and �-actinin into dense actin bundles.

“We think that these radial stress

fi bers then serve as structural templates

to recruit other focal adhesion proteins,”

Gardel explains. “You need a suffi

cient amount of tension to form the

template, but maturation isn’t a tensiondependent

process above that threshold.”

Focal adhesions can be regulated by

tension, however. Cells form smaller adhesions

on soft matrices than they do on

more rigid substrates, suggesting that

ECM stiffness can regulate the assembly

of radial stress fi bers. “That’s something

we’re investigating now,” Gardel says.

“How is stress fi ber assembly regulated at

focal adhesions, and why does that depend

on ECM cues?” Ben Short

1. Oakes, P.W. et al. 2012. J. Cell Biol. http://

dx.doi.org/10.1083/jcb.201107042.

2. Gardel, M.L., et al. 2010. Annu. Rev. Cell Dev.

Biol. 26:315–333.

3. Hotulainen, P., and P. Lappalainen. 2006.

J. Cell Biol. 173:383–394.

4. Choi, C.K., et al. 2008. Nat. Cell Biol.

10:1039–1050.

PHOTO COURTESY OF VENKAT MARUTHAMUTHU

10.1083/jcb.1964iti2jcb.1964iti2Mitch Lesliemitchleslie@comcast.netjcb.1964iti2fig1.epsIn

This IssueNewsAurora B is no Ska fan

By dislodging a microtubule-binding protein, the Aurora B

kinase helps prevent sloppy connections between

chromosomes and the mitotic spindle, Chan et al. suggest.

The KMN complex connects spindle microtubules to kinetochores,

which is essential for chromosomes to separate during

mitosis. To forge solid links between microtubules and the kinetochore,

however, the KMN complex might need help from the Ska

complex, but researchers aren’t sure what recruits this latter group

of proteins to the kinetochore.

Chan et al. found that two members of the KMN complex,

Ndc80 and Mis13, bring the Ska complex to kinetochores. The mitotic

kinase Aurora B inhibited this association by phos phoryl ating the Ska

complex, thereby bumping it from kineto chores. Cells expressing

a non phosphory latable Ska complex formed incorrect kinetochore–

microtubule attach ments and took longer to complete mitosis.

The researchers propose a division of labor during microtubule

attachment. Early on in mitosis, microtubules promiscuously

10.1083/jcb.1972iti3jcb.1972iti3Mitch Lesliemitchleslie@comcast.netjcb.1972iti3fig1.epsIn

This IssueNewsWait, save

that integrin!

SNX17 (green) latches onto

integrin �1 (blue), sparing

it from destruction in the

lysosome.

If you’ve ever rummaged through

the trash to fi nd something you

didn’t mean to toss out, you can

understand one of the problems cells

face. Steinberg et al. reveal that a

sorting protein prevents cells from

throwing away integrins.

Cells continually pluck proteins

from the plasma membrane so they

can be dispatched to the lysosome

for destruction. But some of these

proteins are still useful, so the cell

diverts them back to the plasma membrane. The nexin SNX17 helps

separate the keepers from the trash.

Steinberg et al. devised a new proteomic approach to

determine which proteins SNX17 rescues. They reasoned that

knocking down SNX17 with RNAi should reduce the abundance

of certain proteins because the cell will no longer save these

molecules from destruction. Among the 15 proteins whose levels

declined after RNAi treatment were the �5 and �1 integrins.

Blocking lysosome activity restored the levels of these integrins,

confi rming that without SNX17’s intervention the proteins end

up in the cellular trash.

Because integrins enable cells to get a grip on the extracellular

matrix, the researchers tested whether suppressing SNX17

altered cell movement. In tests of crawling speed, cells depleted

of SNX17 were swifter than normal. It remains to be seen if

the movement of cancer cells—which often swap integrins as

they metastasize—is affected by their ability to spare integrins

from lysosomal destruction. Mitch Leslie

Steinberg, F., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201111121.

connect to and disconnect

from kinetochores. If the Ska

complex fastens to the KMN

complex at this stage, it will

land close to Aurora B and

be phosphorylated, causing it

to drop off. Once the KMN

complex establishes a correctly

oriented connection between a

microtubule and a kinetochore,

the Ska complex can land

without being phosphorylated because tension from the spindle

pulls KMN and the Ska complex away from Aurora B. Ska can

then seal the kinetochore–microtubule link. Delaying the Ska

complex’s arrival until microtubules are correctly attached might

prevent it from clamping weak attachments in place. Mitch Leslie

Chan, Y.W., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201109001.

10.1083/jcb.1965iti1jcb.1965iti1Mitch Lesliemitchleslie@comcast.netjcb.1965iti1fi g1.epsIn This IssueNewsSlow but

steady for NF-�B

Marathon runners

should give NF-�B a

hand. Bakkar et

al.

show that the transcription factor

pushes muscles to make more

mitochondria and to produce the

type of fi ber that tires slowly.

NF-�B has a split

personality during muscle

development and regeneration.

On the one hand, it can be

JCB Highlights 2012 13

Unlike control cells (left), cells that

carry a Ska version that can’t be

phosphorylated by Aurora B (right)

can’t fix incorrect attachments

between microtubules (green) and

kinetochores (red).

Muscle tissue in which the alternative

NF-�B pathway is switched

on (left) harbors more, longer

mitochondria than does control

muscle (right).

activated by the so-called classical pathway that prevents young

muscle cells from differentiating. But NF-�B can also be regulated

by an alternative pathway, which involves a distinct set of proteins,

including IKK� and RelB. Researchers are just starting to investigate

the alternative pathway’s role in muscle. In a previous study, Bakkar et

al. found that it spurs cultured muscle cells to generate mitochondria.

Now, the researchers gauged the alternative pathway’s powers

in vivo by analyzing mice that lacked IKK� or RelB. Muscles from

the animals had fewer mitochondria than normal and showed signs of

an energy shortage. What mitochondria they did contain were

less effi cient. Overexpressing IKK� had the opposite effect, boosting

mitochondrial numbers and power output and inducing muscles to

fashion more slow twitch fi bers. Although weaker than fast twitch

fibers, slow twitch fibers have more stamina and are critical for

long-distance runners.

Bakkar et al. found that the alternative NF-�B pathway exerts

its effects by activating PGC-1�, a master regulator of mitochondrial

biogenesis and function. The researchers also discovered that mTOR,

which responds to hormones, growth factors, and nutrient levels,

activates the pathway. What triggers mTOR to fl ip on NF-�B and

the alternative pathway remains unclear. Mitch Leslie

Bakkar, N., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201108118.

Selected Highlights

10.1083/jcb.1972iti2jcb.1972iti2Mitch Lesliemitchleslie@comcast.netjcb.1972iti2fig1.epsIn

This IssueNewsBRCA1 touches

10.1083/jcb.1964iti1jcb.1964iti1Mitch Lesliemitchleslie@comcast.netjcb.1964iti1fig1.epsIn

This IssueNewsNo crystal

structure? No problem

It’s hard for crystallographers to determine

the structures of all the macromolecular

assemblies that cell biologists want to get

a close look at. But researchers can piece

together precise molecular structures by

The structure of the

Nup84 complex, as

deduced from EM

and domain deletion

mapping results.

10.1083/jcb.1956iti2jcb.1956iti2Ben Shortbshort@rockefeller.edujcb.1956iti2fi g1.epsIn This IssueNewsCUPS provide

up microRNAs

BRCA1, the well-known breast cancer susceptibility

gene, helps control the maturation of microRNAs, Kawai and Amano show. The finding suggests another

way that BRCA1 mutations promote cancer.

The BRCA1 protein helps repair damaged DNA, and mutations

in the gene raise the risk of breast and ovarian cancer and other

tumor types. Some evidence implies that BRCA1 also takes part in

the processing of microRNAs, the short RNA strands that modify

gene expression. For example, microRNA levels are often abnormal

in cells with BRCA1 mutations.

Kawai and Amano tested this idea. They found that boosting

levels of the BRCA1 protein in cell lines increased the levels

of several microRNAs. Deleting BRCA1 from cells had the

opposite effect on the same microRNAs, all of which dwindle

in cancer. BRCA1 had no impact on the levels of microRNAs

considering other readily available data, Fernandez-Martinez et al. suggest.

Their new work builds on their previous

findings that highlighted the value of lowresolution

data, which most labs can obtain fairly

easily but rarely use to infer molecular structures.

For example, with a technique called domain

deletion mapping, which involves pruning

particular proteins and then determining which

interactions remain and which are disrupted,

researchers can deduce the connections between

proteins and their orientations within a complex.

a handle on Acb1 secretion

Bruns et al. describe how several proteins required for

an unconventional secretory pathway gather together in

a novel membrane compartment.

The Acyl-CoA binding protein Acb1 is secreted by starving

budding yeast, but, unlike most secretory proteins, Acb1 doesn’t pass

through the ER and Golgi on its way to the cell surface. The details

of Acb1’s alternative route are unknown, but a diverse set of proteins

mediate its journey. Components required for Acb1 secretion include

autophagy proteins (such as Atg8 and Atg9), proteins that deliver

cargo to multivesicular bodies (for example, Vps23), and Grh1, a

homologue of a mammalian Golgi protein.

Bruns et al. found that, upon starvation, Grh1 concentrated

in membrane compartments near ER exit sites. These structures

didn’t contain markers of the ER, Golgi, or endosomes, but they

did contain Vps23, Atg8, and Atg9, as well as the phosphoinositide

PI(3)P. Blocking PI(3)P synthesis or deleting Grh1 prevented the

formation of these compartments in starving yeast.

whose abundance increases or remains

the same in tumors, however.

BRCA1 bound to primary

microRNA transcripts, Kawai and

Amano found. The protein also interacted

with the DROSHA processing

complex and the proteins Smad3

and p53, which stimulate microRNA

maturation. The results suggest that

BRCA1 promotes the processing

of specifi c microRNAs that might

inhibit tumors. Whether BRCA1’s

Fernandez-Martinez et al. applied the method to the Nup84

complex, which contains seven proteins. Sixteen copies of the

complex form the outer rings of the yeast nuclear pore complex

(NPC). Electron microscopy and domain deletion mapping allowed

the team to obtain spatial restraints, or structural limitations on the

complex’s architecture. Using a computer algorithm, they could

then build possible structures for the Nup84 complex that satisfi ed

these restraints.

Although the analysis confirmed some previous findings,

such as the complex’s overall arrangement and “Y” shape, it also

revealed new features and linked particular functions to specifi c

structures. For example, the fi ndings suggest that three proteins

at the top of the “Y,” Nup85, Nup120, and Seh1, are particularly

important for linking the complex to the rest of the NPC, whereas

Nup120 and a related protein, Nup133, are crucial for stabilizing

the NPC’s interactions with the nuclear envelope. Mitch Leslie

Fernandez-Martinez, J., et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201109008.

By electron microscopy, the

Grh1-containing membranes appeared

cup-shaped, leading the authors to name

them compartments for unconventional

protein secretion or CUPS. Their shape

and the presence of Atg8 and Atg9 are

reminiscent of autophagosome precursors,

but Bruns et al. found that CUPS

weren’t formed in response to the autophagy-inducing

drug rapamycin, suggesting

that CUPS are a novel, albeit

related, compartment. Senior author

BRCA1 (green) interacts with

the DROSHA microRNA

processing complex (red).

effects on microRNAs contribute to its roles in DNA repair remains

to be seen. Mitch Leslie

Kawai, S., and A. Amano. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201110008.

Immunoelectron microscopy

reveals Grh1 (black dots) on

a cup-shaped membrane

structure (arrows, membrane

lamellae).

Vivek Malhotra says that the identifi cation of CUPS gives researchers

a handle to uncover other steps in Acb1 secretion. CUPS may engulf

Acb1 in the cytoplasm and deliver it to the plasma membrane, either

directly or via fusion with secretory endosomes. Ben Short

Bruns, C., et al. 2011. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201106098.

10.1083/jcb.1971ifBen Shortbshort@rockefeller.eduIn FocusNewsBig enough

for two

Study identifies a signaling pathway that may control cell size by linking membrane transport

to mitotic entry.

In order to remain the right size, proliferating

cells must coordinate their

growth and division. If cells divide

before they’ve grown suffi ciently, their

daughters will be too small, whereas cells

may grow too big if division is delayed.

How cells balance growth and division is

unclear, but Anastasia et al. identify a signaling

pathway that might monitor transport

of proteins and lipids to the plasma

membrane and tell yeast cells when they’ve

grown large enough to enter mitosis (1).

jcb.1971iffi g1.epsCell growth requires the delivery of new

material to the plasma membrane via the

secretory pathway. “Membrane growth

must occur at the right place and time during

the cell cycle,” says Doug Kellogg, from the

University of California, Santa Cruz. “Yet

nothing was known about how membrane

traffi cking and the cell cycle are linked.”

Kellogg and colleagues, led by Steph

Anastasia, looked for potential connections

between the two processes by blocking

membrane transport in budding yeast using

a temperature-sensitive mutant of the vesicle

docking protein Sec6 (1). “When we blocked

membrane traffi c, signaling to the core cell

cycle regulators occurred within minutes,”

Kellogg recalls. “The rapidity of that response

really caught our attention.” Specifi -

cally, Anastasia et al. found

that preventing secretion led

to changes in the phosphorylation,

and thus activity,

of Swe1 and Mih1, the

budding yeast homologues

of Wee1 kinase and Cdc25

phosphastase, respectively,

which regulate mitotic entry

in all eukaryotes.

To enter mitosis, Swe1

must be hyperphosphorylated

and inactivated,

whereas Mih1 is dephosphorylated and

switched on. Disrupting membrane transport

reversed these events and caused

yeast to arrest in early mitosis.

Anastasia et al. then looked for proteins

that might regulate Swe1 and Mih1

“This model

argues that

a cell doesn’t

really measure

its size—it

measures the

amount of

growth.”

in response to changes in membrane traffi cking.

PP2A Cdc55 , a phosphatase that controls

both proteins (2, 3), was a good candidate,

and the researchers found that overexpressing

Zds1, a regulator of PP2A Cdc55 , restored

the normal phosphorylation pattern of Swe1

and Mih1 and allowed yeast to divide even

when membrane transport was inhibited.

The authors then traced the signaling

pathway upstream to protein kinase C

(Pkc1), a protein that binds to Zds family

members (4). Pkc1 promoted mitotic entry

by inducing Mih1 dephosphorylation, a

function that was lost in the absence of

PP2A Cdc55 . This suggests that

Pkc1 regulates PP2A Cdc55 ,

though how the kinase does

this remains unknown.

Pkc1, in turn, was regulated

by the GTPase Rho1,

which could induce Mih1

dephosphorylation and mitotic

entry as long as Pkc1

was functional. But how is

this pathway, from Rho1 to

mitotic entry, linked to

membrane traffi c? Inactive

Rho1 is transported into the growing yeast

bud on secretory vesicles and is then activated

by a guanine nucleotide exchange factor

on the plasma membrane (5).

“So we think that it’s the delivery of

Rho1 to the site of membrane growth,

FOCAL POINT

JCB Highlights 2012 15

(Left to right) Tracy MacDonough, Steph Anastasia, Doug Kellogg, Duy Nguyen, and colleagues

(not pictured) uncover a signaling pathway that links membrane transport to mitotic entry in

budding yeast. Cells arrest in early mitosis when signaling through the pathway is disrupted,

either by blocking membrane traffi c or by mutating pathway components like protein kinase C

(right, compared with wild-type yeast, center). The researchers think that the pathway may

allow cells to control their size by coordinating cell growth and division.

and its activation there, that signals that

membrane growth is happening,” explains

Kellogg. According to this model, Rho1

activity and signaling to Swe1 and Mih1

would gradually increase during bud

growth until a critical threshold was

reached that could induce mitotic entry

and cell division. Disruptions to membrane

transport would shut off the pathway

and prevent cells from dividing

when they were too small.

“We’ve all been wondering how a cell

knows how big it is,” says Kellogg. “This

model argues that a cell doesn’t really

measure its size—it measures the amount

of growth that has occurred.” Because all

the components are conserved in higher

eukaryotes, the pathway might operate in

cells of all shapes and sizes.

This “growth-dependent signaling” model

predicts that the strength of the signal is

proportional to membrane growth. Kellogg

and colleagues now want to test this prediction

by developing a biosensor to monitor the

pathway’s activity during the cell cycle and

under different growth conditions. Ben Short

1. Anastasia, S.D., et al. 2012. J. Cell Biol. http://

dx.doi.org/10.1083/jcb.201108108.

2. Pal, G., et al. 2008. J. Cell Biol. 180:931–945.

3. Harvey, S.L., et al. 2011. Mol. Biol. Cell.

22:3595–3608.

4. Drees, B.L., et al. 2001. J. Cell Biol. 154:549–571.

5. Abe, M., et al. 2003. J. Cell Biol. 162:85–97.

PHOTO COURTESY OF VU THAI

Selected Highlights

References

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Kellogg. 2012. A link between mitotic entry and membrane growth suggests a

novel model for cell size control. J. Cell Biol. 197:89–104.

Bakkar, N., K. Ladner, B.D. Canan, S. Liyanarachchi, N.C. Bal, M. Pant,

M. Periasamy, Q. Li, P.M. Janssen, and D.C. Guttridge. 2012. IKKa and

alternative NF-kB regulate PGC-1b to promote oxidative muscle metabolism.

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Bruns, C., J.M. McCaffery, A.J. Curwin, J.M. Duran, and V. Malhotra. 2011.

Biogenesis of a novel compartment for autophagosome-mediated unconventional

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7

Kawai, S., and A. Amano. 2012. BRCA1 regulates microRNA biogenesis via the

DROSHA microprocessor complex. J. Cell Biol. 197:201–208.

Oakes, P.W., Y. Beckham, J. Stricker, and M.L. Gardel. 2012. Tension is required

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JCB Highlights 2012

The extracellular matrix: A dynamic niche

in cancer progression

Pengfei Lu , 1,2,3,4,5 Valerie M. Weaver , 6 and Zena Werb 4,5

The local microenvironment, or niche, of a cancer cell plays

important roles in cancer development. A major component

of the niche is the extracellular matrix (ECM), a complex

network of macromolecules with distinctive physical,

biochemical, and biomechanical properties. Although

tightly controlled during embryonic development and

organ homeostasis, the ECM is commonly deregulated

and becomes disorganized in diseases such as cancer.

Abnormal ECM affects cancer progression by directly promoting

cellular transformation and metastasis. Importantly,

however, ECM anomalies also deregulate behavior of

stromal cells, facilitate tumor-associated angiogenesis and

infl ammation, and thus lead to generation of a tumorigenic

microenvironment. Understanding how ECM composition

and topography are maintained and how their deregulation

infl uences cancer progression may help develop new

therapeutic interventions by targeting the tumor niche.

Introduction

The past 20 years have seen cancer biology and development biology

converge, and both fi elds have greatly benefi ted from each

other’s research progress ( Xie and Abbruzzese, 2003 ; Radtke

and Clevers, 2005 ; Blanpain et al., 2007 ). Retrospectively, such

a convergence is inevitable, as many of the same cell behaviors

and processes essential for embryonic development are also indispensable

for cancer progression ( Egeblad et al., 2010a ). The

concept that local microenvironments, or niches, play an important

role in regulating cell behavior, which is one of the central

themes in classical embryology, has become increasingly accepted

in cancer biology ( Bissell and Radisky, 2001 ; Wiseman

and Werb, 2002 ; Bissell and Labarge, 2005 ).

Correspondence to Zena Werb: zena.werb@ucsf.edu

Abbreviations used in this paper: CAF, cancer-associated fi broblast; LAIR,

leukocyte-associated Ig-like receptor; LOX, lysyl oxidase; MMP, matrix metalloproteinase;

MSC, mesenchymal stem cell.

JCB: Review

1 2 3

Breakthrough Breast Cancer Research Unit, Paterson Institute for Cancer Research, and Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences,

University of Manchester, Manchester M20 4BX, England, UK

4 Department of Anatomy, 5 Developmental and Stem Cell Biology Graduate Program, and 6 Center for Bioengineering and Tissue Regeneration, Department of Surgery,

University of California, San Francisco, San Francisco, CA 94143

Much effort has been devoted to determining how cellular

components of the niche initiate and promote cancer development

( Bhowmick et al., 2004 ). However, recent progress has

also highlighted the importance of noncellular components

of the niche, especially the ECM, during cancer progression

( Sternlicht et al., 1999 ; Paszek et al., 2005 ; Erler et al., 2006 ,

2009 ; Levental et al., 2009 ). Although long viewed as a stable

structure that plays a mainly supportive role in maintaining

tissue morphology, the ECM is an essential part of the milieu of

a cell that is surprisingly dynamic and versatile and infl uences

fundamental aspects of cell biology ( Hynes, 2009 ). Through

direct or indirect means, the ECM regulates almost all cellular

behavior and is indispensable for major developmental processes

( Wiseman et al., 2003 ; Stickens et al., 2004 ; Rebustini

et al., 2009 ; Lu et al., 2011 ).

Consistent with ECM’s many important roles, multiple

regulatory mechanisms exist to ensure that ECM dynamics,

collectively measured by its production, degradation, and remodeling,

are normal during organ development and function

( Page-McCaw et al., 2007 ). Disruption to such control mechanisms

deregulates and disorganizes the ECM, leading to abnormal

behaviors of cells residing in the niche and ultimately failure

of organ homeostasis and function. Indeed, abnormal ECM dynamics

are one of the most ostensible clinical outcomes in diseases

such as tissue fi brosis and cancer ( Cox and Erler, 2011 ).

A major challenge in ECM biology is to understand the

roles of the ECM in normal development and how disruption of

ECM dynamics may contribute to diseases such as cancer. Here,

we examine the diverse properties of the ECM that are essential

for its versatile roles in cancer. We focus on how abnormal ECM

deregulates the behavior of various epithelial and stromal cell

components at different stages of cancer development.

Properties and features of the ECM

The ECM is composed of a large collection of biochemically

distinct components including proteins, glycoproteins, proteoglycans,

and polysaccharides with different physical and biochemical

properties ( Whittaker et al., 2006 ; Ozbek et al., 2010 ).

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The Rockefeller University Press

J. Cell Biol. Vol. 196 No. 4 395–406

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Structurally, these components make up both basement membrane,

which is produced jointly by epithelial, endothelial, and

stromal cells to separate epithelium or endothelium from stroma,

and interstitial matrix, which is primarily made by stromal cells.

Basement membrane is a specialized ECM, which is more compact

and less porous than interstitial matrix. It has a distinctive

composition containing type IV collagen, laminins, fi bronectin,

and linker proteins such as nidogen and entactin, which connect

collagens with other protein components. In contrast, interstitial

matrix is rich in fi brillar collagens, proteoglycans, and various

glycoproteins such as tenascin C and fi bronectin and is thus

highly charged, hydrated, and contributes greatly to the tensile

strength of tissues ( Egeblad et al., 2010b ).

When put together in an orderly manner, the ECM components,

with their remarkable structural and biochemical

diversity and functional versatility, confer upon the matrices

unique physical, biochemical, and biomechanical properties

that are essential for regulating cell behavior. For example, the

physical properties of the ECM refer to its rigidity, porosity,

insolubility, spatial arrangement and orientation (or topography),

and other physical features that together determine its role in

scaffolding to support tissue architecture and integrity. Additionally,

by functioning as a barrier, anchorage site, or movement

track, the ECM’s physical properties play both negative

and positive roles in cell migration ( Fig. 1 , stages 1–3).

In contrast, the biochemical properties of the ECM pertain to

its indirect and direct signaling capabilities that allow cells to sense

and interact with their environments using various signal transduction

cascades emanating from the cell surface to the nucleus,

resulting in gene expression or other changes of cell behavior. For

example, as a highly charged protein network rich in polysaccharide

modifi cations, the ECM can bind to a myriad of growth

factors, including bone morphogenetic proteins, FGFs, hedgehogs,

and WNTs ( Hynes, 2009 ). In so doing, the ECM limits the diffusive

range, accessibility, and signaling direction of ligands to their

cognate receptors ( Fig. 1 , stages 4–6; Norton et al., 2005 ). Additionally,

the ECM can also directly initiate signaling events, particularly

by functioning as a precursor of biologically active signaling

fragments ( Fig. 1 , stage 7; Hynes, 2009 ; Lu et al., 2011 ).

A burgeoning area in ECM biology is how its biomechanical

properties, including the elasticity of the ECM (that

ranges from soft and compliant to stiff and rigid), contribute

to development and disease ( McBeath et al., 2004 ; Reilly and

Engler, 2010 ). As it turns out, ECM elasticity helps determine

how a cell senses and perceives external forces ( Paszek et al.,

2005 ; Lopez et al., 2008 ; Gehler et al., 2009 ) and thus provides

a major environmental cue that determines cell behavior

( Kölsch et al., 2007 ; Montell, 2008 ; Fernandez-Gonzalez

et al., 2009 ; Pouille et al., 2009 ; Solon et al., 2009 ; DuFort

et al., 2011 ). Indeed, the focal adhesion complex, which consists

of integrins and a multicomplex of adaptors and signaling

proteins, can be viewed as a mechanosensor linking the actomyosin

cytoskeleton with the ECM. Many of the focal adhesion

components, including talin and p130Cas, undergo conformational

changes that impart functional consequences in response

to applied force ( Sawada et al., 2006 ; del Rio et al., 2009 ;

Wang et al., 2011 ). Together with the cytoskeleton and nuclear

JCB • VOLUME 196 • NUMBER 4 • 2012

Figure 1. Mechanisms of ECM function. The versatile functions of the ECM

depend on its diverse physical, biochemical, and biomechanical properties.

Anchorage to the basement membrane is essential for various biological

processes, including asymmetric cell division in stem cell biology

and maintenance of tissue polarity (stage 1). Depending on contexts, the

ECM may serve to block or facilitate cell migration (stages 2 and 3). In

addition, by binding to growth factor signaling molecules and preventing

their otherwise free diffusion, the ECM acts as a sink for these signals and

helps shape a concentration gradient (stage 4). Certain ECM components,

including heparan sulfate proteoglycans and the hyaluronic acid receptor

CD44, can selectively bind to different growth factors and function as a

signal coreceptor (stage 5) or a presenter (stage 6) and help determine

the direction of cell–cell communication ( Lu et al., 2011 ). The ECM also

direct signals to the cell by using its endogenous growth factor domains

(not depicted) or functional fragment derivatives after being processed by

proteases such as MMPs (stage 7). Finally, cells directly sense the biomechanical

properties of the ECM, including its stiffness, and change a wide

variety of behaviors accordingly (stage 8).

matrices, nuclear envelope, and chromatin, they constitute a sophisticated

mechanosensing machinery that determines how

cells react to forces from the ECM ( DuFort et al., 2011 ). Interestingly,

however, changes in mechanical force can be converted

into differences in TGF- � signaling activities in the mouse tendon

( Maeda et al., 2011 ), suggesting that conventional signaling

pathways can be used to interpret the biomechanical properties

of the ECM. As a result, ECM’s biomechanical properties regulate

various essential cell behaviors, including cell fate determination,

differentiation, and tissue function ( Fig. 1 , stage 8;

Engler et al., 2006 ; Lutolf et al., 2009 ; Gilbert et al., 2010 ).

Importantly, several outstanding characteristics of the

properties of the ECM contribute to its importance in development

and disease. First, the different properties of the ECM are

not independent; rather, they are intertwined. Therefore, when

the ECM stiffens, as, for example, under pathological conditions,

its biomechanical properties change, and cells respond by

exerting markedly different kinds of force ( Yu et al., 2011 ). In

addition, matrix stiffening also changes other ECM physical

properties and, as a consequence, directly impacts how migrating

cells interact with the ECM. Thus, linearized cross-linked

collagen bundles, which are quite stiff, potentiate cell migration,

whereas a dense network of stiff cross-linked matrix fi bers impedes

migration, unless matrix metalloproteinases (MMPs) are

simultaneously activated ( Egeblad et al., 2010b ).

Second, the ECM is highly dynamic, constantly being

remodeled in different tissues at various embryonic and postnatal

stages. ECM dynamics may result from changes of the absolute

amount or composition of the ECM, for example as a

result of altered synthesis or degradation of one or more

ECM components. Alternatively, ECM dynamics may show

no compositional changes of its components but instead involve

only how individual ECM components are laid down, crosslinked,

and spatially arranged together via covalent and noncovalent

modifi cations.

Finally, one of the most prominent features of cell–ECM

interactions is that they are reciprocal. On the one hand, cells

are constantly creating, breaking down, or otherwise rearranging

and realigning ECM components to change one or more

properties of the ECM. On the other hand, because the ECM

regulates diverse cell behavior, any changes in the ECM as a

result of cellular activities will in turn infl uence adjacent cells

and modify their behaviors ( Butcher et al., 2009 ). This feedback

regulatory mechanism between cells and the ECM allows

cells and tissues to swiftly adapt to their environment

( Samuel et al., 2011 ).

Deregulated ECM dynamics are a hallmark

of cancer

ECM remodeling is tightly regulated during development and

primarily accomplished by controlling the expression or activities

of ECM enzymes at multiple levels. Take for example ECM

degrading enzymes, which include MMPs, a disintegrin and

metalloproteinase with thrombospondin motifs, and the serine

protease plasmin: left unchecked, the potent activities of these

enzymes can have devastating destructive consequences on tissues

and cause demise of the whole organism. As a result, ECM

remodeling enzymes are not only regulated at the transcriptional

and translational levels but also posttranslationally with

the use of their functionally inhibitive prodomains and selective

proteinase inhibitors ( Page-McCaw et al., 2007 ; Aitken and

Bägli, 2009 ).

Despite having multiple control mechanisms, activities

of ECM remodeling enzymes may be deregulated with age or

under disease conditions. Consequently, ECM dynamics may

become abnormal as the amount, composition, or topography of

the ECM turn aberrant, leading to disorganization and changes

in the essential properties of the ECM. The main contributors

of altered activities of ECM remodeling enzymes and thus

abnormal ECM metabolism are stromal cells, including cancerassociated

fi broblasts (CAFs) and immune cells ( Bhowmick

et al., 2004 ; Orimo et al., 2005 ). However, other cell types, including

epithelial cells and mesenchymal stem cells (MSCs),

may also be involved at late stages of cancer development

( Quante et al., 2011 ; Singer and Caplan, 2011 ).

Abnormal ECM dynamics are well documented in clinical

studies of many diseases and are a hallmark of cancer. For

example, excess ECM production or reduced ECM turnover are

prominent in tissue fi brosis of many organs ( Frantz et al., 2010 ).

Various collagens, including collagen I, II, III, V, and IX, show

increased deposition during tumor formation ( Zhu et al., 1995 ;

Kauppila et al., 1998 ; Huijbers et al., 2010 ). As we age, there is

a reduction of collagen deposition and increased MMP activity

( Norton et al., 2005 ; Butcher et al., 2009 ). Moreover, many

JCB Highlights 2012 19

other ECM components and their receptors such as heparan sulfate

proteoglycans and CD44 that facilitate growth factor signaling

are frequently overproduced in cancer ( Kainz et al.,

1995 ; Stauder et al., 1995 ; Nasser, 2008 ). Thus, abnormal

changes in the amount and composition of the ECM can greatly

alter ECM biochemical properties, potentiate the oncogenic

effects of various growth factor signaling pathways, and deregulate

cell behaviors during malignant transformation.

In addition to changes in its biochemical properties, the

architecture and other physical properties of tumor-associated

ECM are fundamentally different from that of the normal tissue

stroma; rather than relaxed nonoriented fi brils, the collagen I in

breast tumors is often highly linearized and either oriented adjacent

to the epithelium or projecting perpendicularly into the

tissue ( Provenzano et al., 2006 ; Levental et al., 2009 ). Consistent

with these changes, expression of many ECM remodeling

enzymes is often deregulated in human cancers. Heparanases,

6-O-sulfatases, cysteine cathepsins, urokinase, and, most notably,

many MMPs are frequently overexpressed in different cancers

( Ilan et al., 2006 ; Kessenbrock et al., 2010 ).

Furthermore, ECM’s biomechanical properties also change

under disease conditions. For example, tumor stroma is typically

stiffer than normal stroma; in the case of breast cancer, diseased

tissue can be 10 times stiffer than normal breast ( Levental et al.,

2009 ; Lopez et al., 2011 ). Part of the increase in tissue stiffness

can be attributed to excess activities of lysyl oxidase (LOX),

which cross-links collagen fi bers and other ECM components.

Indeed, up-regulation of LOX expression has been observed

in various cancers, including breast cancer and head and neck

cancer, and is a poor prognostic marker ( Le et al., 2009 ; Barker

et al., 2011 ). Importantly, a study using mouse genetics has

shown that overexpression of LOX increases ECM stiffness and

promotes tumor cell invasion and progression ( Levental et al.,

2009 ). In contrast, inhibition of LOX reduces tissue fi brosis and

tumor incidence in the Neu breast cancer model ( Levental et al.,

2009 ). Together, these data demonstrate that deregulation of

collagen cross-linking and ECM stiffness is more than just a secondary

outcome but instead plays a causative role in cancer pathogenesis.

Interestingly, however, overexpression of LOX alone

is insuffi cient to cause tumors to form ( Levental et al., 2009 ), suggesting

that deregulation of ECM remodeling is a coconspirator

rather than a primary inducer of tumorigenesis in the breast.

Abnormal ECM dynamics during

cancer progression

Multicellular organisms have evolved many redundant mechanisms

to prevent a cell that is intimately integrated with other cells

in a functional tissue from becoming cancerous and leading to

organ failure and demise of the organism. To overcome these protective

measures and become cancerous, a cell must accumulate

multiple oncogenic properties that ultimately result in malignant

transformation. These include the acquisition by cancer cells of the

ability to survive, grow, and invade ( Hanahan and Weinberg, 2000 ,

2011 ). Along the way, cancer cells often lose their differentiation

state and polarity, disrupt tissue integrity, and corrupt stromal cells

to promote their own growth at both primary tumor and distant

sites ( Feigin and Muthuswamy, 2009 ; Luo et al., 2009 ).

Extracellular matrix in cancer progression • Lu et al.

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398

Abnormal ECM can promote many of the aforementioned

steps. An increase in collagen deposition or ECM stiffness,

alone or in combination, up-regulates integrin signaling and can

thus promote cell survival and proliferation ( Wozniak et al.,

2003 ; Paszek et al., 2005 ). Increased collagen cross-linking and

ECM stiffness as a result of LOX overproduction promote focal

adhesion assembly and ERK and PI3 kinase signaling and facilitate

Neu-mediated oncogenic transformation ( Levental et al.,

2009 ). Moreover, various ECM components or their functional

fragment derivatives have pro- or antiapoptotic effects ( Mott

and Werb, 2004 ). Therefore, deregulation of ECM remodeling

can lead to apoptotic evasion by mutant cells. Among the numerous

roles of abnormal ECM, we focus in the next section on

how it may convert a normal stem cell niche into a cancer stem

cell niche and how it may disrupt tissue polarity and integrity to

promote tissue invasion, both of which are essential steps during

cancer progression.

The ECM is an essential component

of the stem cell niche and the cancer

stem cell niche

Mounting evidence suggests that the ECM is an essential noncellular

component of the adult stem cell niche. For example,

various ECM receptors have been used as markers to enrich

adult stem cells in many in vitro and in vivo systems ( Shen

et al., 2008 ; Raymond et al., 2009 ), suggesting that contact with

the ECM is necessary for cells to acquire or maintain stem cell

properties. In contrast, loss of ECM contact by either functional

ablation ( Yamashita et al., 2005 ; Tanentzapf et al., 2007 ;

O’Reilly et al., 2008 ) or reduction ( Frye et al., 2003 ) of the

ECM receptor integrins or reduction of ECM components, including

the glycoproteins osteopontin ( Kollet et al., 2006 ; Lymperi

et al., 2010 ), tenascin C ( Garcion et al., 2004 ), or biglycan

( Bi et al., 2007 ), reduces the number of stem cells in different

vertebrate and invertebrate systems.

Studies now show that the ECM plays multiple roles in the

stem cell niche. For example, ECM receptors allow stem cells to

anchor to the special local niche environment where stem cell

properties can be maintained. Such an anchorage physically constrains

stem cells to make direct contact with niche cells, which

produce paracrine signaling molecules that are essential for

maintaining stem cell properties ( Fig. 2 A , stage 1; Li and Xie,

2005 ). Moreover, anchorage allows stem cells to maintain cell

polarity, orient their mitotic spindles, and undergo asymmetric

cell division ( Fig. 2 A , stage 2), a fundamental mechanism

whereby stem cell self-renewal and differentiation are thought to

be determined ( Lambert and Nagy, 2002 ; Fuchs et al., 2004 ;

Lechler and Fuchs, 2005 ; Yamashita and Fuller, 2008 ).

In addition to maintaining stem cell properties, the ECM,

via its diverse and potent signaling abilities, can directly regulate

stem cell differentiation, although the molecular details

of how this is achieved have only just started to emerge. Many

of the signaling pathways that play an important role in stem

cell biology in numerous model systems are subject to ECM

modulation. For example, tenascin C can modulate FGF2 and

BMP4 signaling, both of which are essential for neural stem cell

biology ( Garcion et al., 2004 ), whereas the ECM regulates

JCB • VOLUME 196 • NUMBER 4 • 2012

Figure 2. ECM is an essential component of normal and cancer stem cell

niche. The ECM plays multiple roles in maintaining stem cell properties.

(A) ECM anchorage restricts stem cells in the niche and thus allows them to be

exposed to paracrine (stage 1) and cell–cell contact signals (not depicted)

that are essential for maintaining stem cell properties. Anchorage is also

important for orienting the mitotic spindle and makes it possible for stem cells

to undergo asymmetric cell division (stage 2), which is essential for stem

cell self-renewal and generation of daughter cells that are destined to

undergo cell differentiation. The exact mechanism whereby ECM anchorage

controls asymmetric cell division remains unclear, although one possibility

is to allow cytoplasmic cell fate determinants to be differentially distributed

between the daughter cells. The ECM also maintains stem cell properties

via its many other features including its biomechanical properties such as

ECM stiffness that affects cell fate determination (stage 3). (B) In the presence

of abnormal ECM (pink) or loss of ECM contact, stem cell properties

fail to be maintained and undergo symmetric cell division instead, leading

to an overexpansion of the (cancer) stem cell pool. Abnormal changes of

the ECM can also disrupt the cellular differentiation process, resulting in

loss of differentiation and an increase of stem/progenitor cells.

ligand accessibility of the Janus kinase–signal transducer and

activator of transcription signaling pathway in the fl y testis

( Yamashita et al., 2005 ).

The biomechanical properties of the ECM also play an

important role in regulating stem cell biology. MSCs grown on

polymer gels with similar elasticity to the brain express neuronal

markers and morphology, whereas those grown on gels that

are semicompliant like smooth and skeletal muscle tissues or

rigid like the bone express muscle or bone proteins, respectively

( McBeath et al., 2004 ; Engler et al., 2006 ). Likewise, muscle

stem cells grown on soft hydrogels with elasticity mimicking

that of real muscle differentiate into functional muscle ( Gilbert

et al., 2010 ), highlighting the great promise that tissue engineering

may hold in regenerative medicine. Together, it is conceivable

that by modulating various aspects of ECM properties, a

lineage-specifi c ECM may be created to facilitate cell differentiation

processes during lineage specifi cation and organ development

( Fig. 2 A , stage 3).

The decision between stem cell expansion and differentiation

is a delicate one and must be tightly controlled during normal

organ homeostasis and function. An imbalance of these two

events can lead to the generation of tumor-initiating cells, which

have been called cancer stem cells by either overexpanding the

stem cell pool or a failure in stem cell differentiation. Indeed,

loss of cell polarity as a result of ablation of Numb or Lgl protein,

essential components of the cell polarity machinery, disrupts

asymmetric cell division and leads to overexpansion of

neural stem cells and tumor formation in the brain ( Li et al.,

2003 ; Klezovitch et al., 2004 ). Therefore, the essential roles

that the ECM plays in the stem cell niche make it a likely candidate

to be targeted to create a cancer stem cell niche during cellular

transformation. It is possible, at least theoretically, that

deregulated ECM dynamics may cause formation of abnormal

lineage-specifi c ECM and lead to cancer stem cell overexpansion

and loss of differentiation ( Fig. 2 B ). However, whether a

cancer stem cell niche may result from such an event of ECM

dynamics deregulation remains to be rigorously tested.

The ECM maintains tissue polarity

and architecture and prevents cancer

cell invasion

An important feature of epithelial organs, which is often lost in

cancer, is that cells in them have distinct polarity and architecture

that are indispensable for organ formation and function ( Ghajar

and Bissell, 2008 ). Studies have shown that ECM is essential

for the establishment and maintenance of tissue polarity and architecture.

For example, � 1-integrin maintains tissue polarity in

solid organs including the mammary gland ( Akhtar et al., 2009 ),

whereas various ECM components are important for planar cell

polarity during epithelial morphogenesis ( Davidson et al., 2006 ;

Latimer and Jessen, 2010 ; Skoglund and Keller, 2010 ). Abnormal

ECM dynamics can compromise basement membrane as a

physical barrier and promote epithelial–mesenchymal transition,

which together can facilitate tissue invasion by cancer cells

( Song et al., 2000 ; Duong and Erickson, 2004 ; Radisky and

Radisky, 2010 ).

One way the physical barrier of basement membrane can

be removed, at least partially, is by overexpressing MMPs. Consistent

with this notion, mice overexpressing MMP3, MMP7, or

MMP14 form mammary tumors ( Sternlicht et al., 1999 ). It is

reasonable to predict that cancer cells or their accompanying

stromal and immune cells bearing MMPs have selective advantage

over those that are not because, presumably, they can readily

enter and exit the endothelial basement membrane and

metastasize to distant sites. Additionally, changes in ECM topography

may also facilitate cancer cell migration. Thickening

and linearization of collagen fi bers are common in cancers, and

JCB Highlights 2012 21

they are often found in areas where active tissue invasion and

tumor vasculature are observed ( Condeelis and Segall, 2003 ;

Provenzano et al., 2006 ; Levental et al., 2009 ), suggesting that they

play an active role in facilitating cancer cell invasion. Indeed,

studies using live imaging have shown that cancer cells migrate

rapidly on collagen fi bers in areas enriched in collagen ( Wang

et al., 2002 ; Condeelis and Segall, 2003 ; Wyckoff et al., 2007 ).

Together, deregulation of ECM dynamics can facilitate cellular

dedifferentiation and cancer stem cell expansion. Additionally,

they disrupt tissue polarity and promote tissue invasion. As a

result, epithelial cells are directly affected by deregulated ECM

dynamics, leading to cellular transformation and metastasis.

Abnormal ECM promotes formation of a

tumor microenvironment

Abnormal ECM also indirectly affects cancer cells by infl uencing

the behavior of stromal cells, including endothelial cells, immune

cells, and fi broblasts, which are the main initial culprits

that cause abnormal ECM production ( Bhowmick et al., 2004 ;

Orimo et al., 2005 ; Quante et al., 2011 ). As a result, abnormal

ECM further perpetuates the local niche and promotes the formation

of a tumorigenic microenvironment.

Role of the ECM in tumor angiogenesis

and lymphangiogenesis

As a disorganized organ, tumor develops by using many of the

same cellular and developmental processes essential for organogenesis

( Ruoslahti, 2002 ; Egeblad et al., 2010a ). For a tumor

to increase in size, for example, tumor cells face the same increasing

demand for nutrient, oxygen, and waste exchange as

normal cells do in a growing organ during development. As in

normal development, such a demand is met by angiogenesis, the

process whereby new blood vessels sprout from the existing

vasculature ( Davis and Senger, 2005 ). Furthermore, tumor vasculature,

together with the lymphatic system, is the main route

through which cancer cells metastasize and immune cells infi ltrate.

Consequently, tumor-associated angiogenesis and lymphangiogenesis,

the process whereby lymphatic vessels are

generated, are important aspects of cancer progression ( Fig. 3 ;

Avraamides et al., 2008 ).

The role of abnormal ECM in tumor angiogenesis is a result

of the various functions that ECM components play in

blood vessel formation during normal development. For example,

many ECM fragments, including endostatin, tumstatin,

canstatin, arresten, and hexastatin, all of which are derived from

collagens type IV and XVIII, have potent stimulatory or inhibitory

effects on angiogenesis ( Mott and Werb, 2004 ). They are

likely to collaborate with other pro- or antiangiogenic factors,

including VEGF, to determine where to initiate vascular branching

and the fi nal branch pattern ( Fig. 3 A , stage 1). To initiate

vascular branching, vessel basement membrane ECM needs to

be removed most likely by MMPs expressed by invading endothelial

cells ( Fig. 3 A , stage 2). MMPs, for example MMP14

(MT1-MMP), are also required for the invading tip cell, which

is at the leading edge of an endothelial branch, to wade through

the interstitial matrix toward target cells ( Fig. 3 A , stage 3;

Genís et al., 2007 ; van Hinsbergh and Koolwijk, 2008 ). In addition,

Extracellular matrix in cancer progression • Lu et al.

399

22

400

Figure 3. ECM role in tumor angiogenesis, lymphangiogenesis, and

infl ammation. (A) Angiogenesis and lymphangiogenesis depend on the

ECM. Tumor cells produce various components, including VEGF and angiogenic

and antiangiogenic ECM fragments, to regulate blood vessel

formation (stage 1). During branch initiation, endothelial cells secrete proteases

to break down the basement membrane to grow out (stage 2). The

outgrowth process of endothelial branching is propelled by at least two

groups of cells: tip cells, which lead the migration toward the angiogenic

chemoattractant source, and stalk cells, which depend on the ECM and

its derivatives to survive and proliferate to provide building blocks for vessel

formation (stage 3). Additionally, ECM components participate in cell

migration and other aspects of tubulogenesis of blood vessels. Although

details remain unclear, lymphangiogenesis depends on the ECM and, together

with angiogenesis, provides routes for cancer cell metastasis and

immune cell infi ltration. (B) The ECM plays multiple roles in tumor infl ammation.

In addition to promoting survival and proliferation (not depicted),

ECM components function as a chemoattractant to immune cells (stage a).

The exact details of how immune cells including neutrophil transmigrate

endothelial basement membrane are not clear, though it seems the ECM

plays both positive and negative roles in the process. Macrophage

activation depends on the ECM to release its potent cytokine signals

and protease content (stage b). Further, immune cell differentiation, including

maturation of T helper cells, requires participation of ECM components

(stage c).

hypoxia can lead to overproduction of LOX-like protein-2 and

a subsequent increase in ECM cross-linking and stiffening,

resulting in sprouting angiogenesis ( Bignon et al., 2011 ). These

data suggest that ECM biomechanical properties also play

essential roles in angiogenesis.

Angiogenesis is a complex process, requiring coordination

of many cellular activities. Thus, in addition to guiding endothelial

cell migration and branching, ECM and its fragments may be

involved in endothelial cell survival and proliferation to supply

JCB • VOLUME 196 • NUMBER 4 • 2012

cellular building blocks for vessel growth ( Sweet et al., 2011 ).

Furthermore, ECM components are involved in cellular morphogenesis,

including vessel lumen formation ( Newman et al., 2011 )

and other aspects of tubulogenesis during tumor angiogenesis

( Davis and Senger, 2005 ). The biomechanical properties of the

ECM appear to play an especially important role in this process.

Indeed, vascular networks with markedly distinct branching patterns

have been observed when endothelial cells are grown on

matrix with different elasticity ( Myers et al., 2011 ).

Finally, new ECM is deposited to form basement membrane

to surround blood vessels during tumor angiogenesis.

Importantly, however, the basement membrane of the tumor

vasculature is more porous and leaky than normal ( Hewitt et al.,

1997 ; Hashizume et al., 2000 ), which facilitates tumor cell metastasis

and immune cell infi ltration and promotes cancer progression

( Ruoslahti, 2002 ; Egeblad et al., 2010a ). Likewise, the

lymphatic system can also transport tumor and immune cells.

Recent studies show that the ECM receptor integrin � 9 � 1 plays

an important role in the formation of lymphatic vessels ( Huang

et al., 2000 ; Avraamides et al., 2008 ), suggesting that the ECM

is likely to play a role in tumor lymphangiogenesis as well.

However, this suggestion awaits further experimental testing,

as do the details of how abnormal ECM dynamics may deregulate

lymphangiogenesis during cancer progression.

Role of the ECM in tumorassociated

infl ammation

Infl ammation, characterized by massive infl ux of immune cells,

plays a causative role in cancer development. Although their

initial function is supposed to suppress tumor growth, immune

cells including macrophages are often altered and recruited by

tumor cells at later stages to promote cancer ( Coussens and

Werb, 2002 ). As in tumor angiogenesis, abnormal ECM affects

many aspects of immune cell behaviors, including infi ltration,

differentiation, and functional activation.

For example, mice lacking the ECM glycoprotein SPARC

(secreted protein acidic and rich in cysteine) have an increased

number of macrophages in tumors, suggesting that the ECM

can infl uence the number of immune cells. One way the ECM

affects immune cells is by regulating cell proliferation ( Adair-

Kirk and Senior, 2008 ; Sorokin, 2010 ). ECM components also

may function as chemoattractants to immune cells ( Fig. 3 B ,

stage a). For example, elastin fragments are able to recruit

monocytes, but not neutrophils, in the rat lung ( Houghton et al.,

2006 ). The acetylated tripeptide Pro-Gly-Pro derived from collagen

I proteolysis by MMP8 or MMP9 can functionally mimic

the chemoattractant CXCL8 on neutrophils in a lung infl ammation

model ( Weathington et al., 2006 ). Alternatively, activation

of collagen receptor DDR1 can also promote macrophage infi ltration

in atherosclerotic plaques ( Franco et al., 2009 ).

To reach the infl amed or tumor sites, immune cells encounter

two kinds of potential ECM barriers: the endothelial

basement membrane and interstitial matrix. Studies using EM

and, more recently, intravital imaging have shown that transmigration

across the endothelial basement membrane is a ratelimiting

step during T cell extravasation ( Wang et al., 2006 ;

Bartholomäus et al., 2009 ). Interestingly, however, inhibition of

integrin � 6 � 1, which binds to laminin, results in reduced neutrophil

infi ltration and trapping of neutrophils between endothelium

and the basement membrane ( Dangerfi eld et al., 2002 ).

These data suggest that, although the basement membrane is a

barrier to immune cell extravasation, binding and attachment to

ECM components are necessary for transmigration to occur. It

remains unclear how immune cells transmigrate across the basement

membrane, for example, regarding whether ECM degradation

is involved and whether immune cells have preferred and

presumably more porous passage sites along the vessel wall

( Rowe and Weiss, 2008 ). Once they enter the stroma, immune

cells travel through the interstitial matrix during infi ltration. As

in the cases of tumor and endothelial cells, ECM topography

such as collagen fi bril size and density can infl uence migration

of immune cells ( Fig. 3 B , stage a; Lämmermann et al., 2008 ).

The ECM also regulates the activation of immune cells.

For example, increased ECM stiffness can promote integrinmediated

adhesion complex assembly and activate T cells

( Ashkar et al., 2000 ; Adler et al., 2001 ; Hur et al., 2007 ; Sorokin,

2010 ). Although collagen type I promotes infi ltration of immune

cells, it inhibits the ability of macrophages to kill cancer

cells by blocking polarization and, thus, activation of macrophages

( Fig. 3 B , stage b; Kaplan, 1983 ). These results highlight

the complex nature of how ECM deregulation may affect

behaviors of different groups of immune cells. The inhibitory

effect of collagen I on immune cells is likely mediated by its

binding with the leukocyte-associated Ig-like receptors (LAIRs),

which are expressed at the surface of most immune cells

( Meyaard, 2008 ; Frantz et al., 2010 ). At present, it is not clear

whether LAIRs and integrins cooperate; however, the activation

of LAIRs is a plausible mechanism whereby high levels of

tumor collagen can attenuate the otherwise tumor-suppressive

function of immune cells. Additionally, the ECM plays an important

role in immune cell differentiation, including the maturation

process of T helper cells ( Chabas et al., 2001 ; Hur et al., 2007 ). A

study also shows that hyaluronan can induce regulatory T cell differentiation

from effector memory T cell precursors ( Bollyky

et al., 2011 ). Therefore, one plausible mechanism whereby abnormal

ECM sabotages the immune system during cancer development

may be to prevent immune cells from undergoing their

normal differentiation and maturation process ( Fig. 3 B , stage c).

Finally, another group of stromal cells, MSCs, has

emerged as an important player in the cancer niche. As multipotent

stem cells, MSCs normally can give rise to various cell

types, including osteoblasts, chondrocytes, adipocytes, and, at

least under pathological conditions, CAFs ( Quante et al., 2011 ),

which are essential for abnormal ECM metabolism. Because the

ECM plays an important role in MSC differentiation ( Engler et al.,

2006 ), it is likely that MSCs may be yet another target cell population

of abnormal ECM dynamics in the formation of a cancer

niche. This is an especially important point, as MSCs can exert

pleiotropic effects on infl ammation ( Aggarwal and Pittenger,

2005 ; Ripoll et al., 2011 ; Singer and Caplan, 2011 ). Together,

these data reinforce the possibility that, once beyond a certain

threshold, deregulated ECM dynamics may cause irreversible

changes to the normal niche and convert it into a cancerpromoting

environment.

JCB Highlights 2012 23

In summary, abnormal ECM dynamics deregulate behaviors

of both cancer cells and stromal cells. On the one hand,

ECM anomalies promote cancer cell transformation and tissue

invasion; on the other hand, they help generate a tumorigenic

niche that further facilitates cancer progression. Such a doublewhammy

effect is a recurring theme at later stages of cancer

metastasis, as is evident from the next section.

The ECM: An essential component of

premetastatic and metastatic niches

Cancer cell metastasis is a multistep process, consisting of local

invasion and intravasation at the primary site, survival in the

circulation, and extravasation and colonization at the distant

site ( Paget, 1889 ). Depending on cancer type and organ destination,

these steps may have distinct kinetics during cancer metastasis

( Nguyen et al., 2009 ). A successful metastasis requires not

only a local niche to support cancer cell growth at the primary

site but also one, the metastatic niche, to allow invading cancer

cells to survive, colonize, and expand to form a macrometastasis

( Psaila and Lyden, 2009 ).

Although still in its infancy, studies support that the ECM

is, as in the primary tumor niche, an essential component of the

metastatic niche. For example, although most metastatic cancer

cells die, mammary carcinoma cells expressing the hyaluronan

receptor CD44 survive better than cells with low levels of CD44

( Yu et al., 1997 ). These data imply that hyaluronan and maybe

other ECM components promote survival of metastatic cancer

cells. Moreover, as in the case of primary tumor niche, LOX activities

are often up-regulated in metastatic cancer sites as a result

of increased production from cancer cells or activated

fi broblasts at the metastatic niche ( Erler et al., 2009 ). Increase in

mechanical force as a result of LOX expression and ECM stiffening

presumably facilitates colonization of cancer cells and infi

ltration of immune cells at the metastatic site. These changes

may be similar to the ones at the primary niche and together may

further trigger the angiogenic switch and lead to cancer cell expansion

from micrometastasis to macrometastasis ( Fig. 4 ). However,

this notion remains to be tested experimentally.

Remarkably, mounting evidence suggests that cancer cells

may remotely modify, often with the involvement of other cell

types including hematopoietic progenitor cells, distant sites and

proactively participate in the creation of a premetastatic niche

before metastasis ( McAllister and Weinberg, 2010 ; Bateman,

2011 ). For example, cancer cells at the primary site produce

osteopontin and other factors to recruit granulin-expressing

hematopoietic progenitor cells, which can then deregulate

behaviors of the distant stromal cells ( Elkabets et al., 2011 ).

Interestingly, granulin, belonging to the epithelin family of

secreted growth factors, can increase the expression of a variety

of ECM components and their modifying enzymes in stromal

fi broblasts ( Elkabets et al., 2011 ).

Changes of ECM composition are important for continued

recruitment of hematopoietic progenitor cells to the premetastatic

niche. For example, increased fi bronectin expression

is essential for VEGF receptor 1 + (VEGFR1 + ) hematopoietic

progenitor cells, which also express the fi bronectin receptor

integrin � 4 � 1, to migrate and adhere to the niche in the lung

Extracellular matrix in cancer progression • Lu et al.

401

24

402

Figure 4. Abnormal ECM promotes cancer progression. (A) ECM remodeling is tightly controlled to ensure organ homeostasis and functions. Normal ECM

dynamics are essential for maintaining tissue integrity and keep rare tumor-prone cells, together with resident fi broblasts, eosinophils, macrophages, and

other stromal cells, in check by maintaining an overall healthy microenvironment. (B) With age or under pathological conditions, tissues can enter a series

of tumorigenic events. One of the earlier events is the generation of activated fi broblasts or CAFs (stage 1), which contributes to abnormal ECM buildup

and deregulated expression of ECM remodeling enzymes (stage 2). Abnormal ECM has profound impacts on surrounding cells, including epithelial,

endothelial, and immune cells and other stromal cell types. Deregulated ECM promotes epithelial cellular transformation and hyperplasia (stage 3).

(C) In late-stage tumors, immune cells are often recruited to the tumor site to promote cancer progression (stage 4). In addition, deregulated ECM affects various

aspects of vascular biology and promotes tumor-associated angiogenesis (stage 5). Creation of a leaky tumor vasculature in turn facilitates tumor cell

invasion and metastasis to distant sites (stage 6). (D) At distant sites, cancer cells leave the circulation and take hold of the local tissue. Together with local

stromal cells, cancer cells express ECM remodeling enzymes and create a local metastatic niche. Abnormal niche ECM promotes extravasation, survival,

and proliferation of cancer cells (stage 7). At later stages when cancer cells awake from dormancy, abnormal ECM turns on the angiogenic switch (stage 8),

presumably using a mechanism similar to that used at the primary site (stage 5), and promotes the rapid growth of cancer cells and an expansion of

micrometastasis to macrometastasis.

( Kaplan et al., 2005 ). Once there, VEGFR1 + hematopoietic progenitor

cells secrete MMP9, which is known to play a role in

lung-specifi c metastasis ( Hiratsuka et al., 2002 ), and thus further

modulate and deregulate the premetastatic niche. In addition

to fi bronectin, other ECM components may also be important

for the function of the premetastatic niche. For example, hyaluronan

and its receptor CD44 facilitate signaling via C-X-C chemokine

receptor 4 (CXCR4) and its ligand stromal-derived growth

factor 1 (SDF1/CXCL12; Netelenbos et al., 2002 ; Avigdor et al.,

2004 ), which are essential for organ-specifi c metastasis of

tumor cells to the lung or bone marrow ( Jones et al., 2006 ).

Thus, these data suggest that deregulation of ECM dynamics is

an important step during the formation of a premetastatic niche.

Collectively, a picture has started to emerge with regard

to ECM’s roles in cancer progression: normal ECM dynamics

are essential for embryonic organ development and postnatal

function ( Fig. 4 A ); deregulated ECM dynamics disrupt tissue

polarity, architecture, and integrity and promote epithelial cell

transformation and invasion ( Fig. 4 B ). Furthermore, abnormal

ECM dynamics derail stromal cell behavior, leading to

tumor-promoting angiogenesis and infl ammation by endothelial

cells and immune cells, respectively, both at the primary

JCB • VOLUME 196 • NUMBER 4 • 2012

and metastatic sites. The resultant changes in the stromal components

further exacerbate the tumorigenic microenvironment

and facilitate the process of oncogenic transformation, tissue

invasion, and metastasis during cancer initiation and progression

( Fig. 4, C and D ).

Concluding remarks

From the initial belief that the intrinsic properties of cancer

cells determine most major aspects of cancer initiation and progression,

our understanding of cancer biology has taken remarkable

strides. We now regard cancer as a heterogeneous disease

not only in the sense that different molecular etiologies may underlie

the same clinical outcome but also that multiple cell

types, in addition to cancer cells, and noncellular components

need to be mobilized and coordinated to support the survival,

growth, and invasion of cancer cells. As a major component of

the local niche, the ECM has emerged as an essential player at

various stages of the carcinogenic process. Its functional diversity

and dynamic nature, which allows the ECM to be an active

participant in most major cell behavior and developmental processes,

also makes it a necessary target whose deregulation may

be a rate-limiting step in cancer progression.

An important area of future cancer research will be to determine

whether abnormal ECM could be an effective cancer therapeutic

target. To achieve this goal, we must understand how ECM

composition and organization are normally maintained and regulated

and how they may be deregulated in cancer. A daunting task

in this regard will be to determine the kind of ECM changes that

have causative effects on disease progression and how these

changes of the ECM, alone or in combination, may affect cancer

cells and cells in the stromal compartment. Additionally, with the

growing documentation of the diverse functions of the ECM in

development and cancer, a major challenge will be to understand

the molecular basis of these functions, whether they involve only

receptor signaling, rearrangements of the cytoskeleton, changes

of gene expression, or other aspects of cell behavior, and how

such changes are integrated with conventional signaling cascades

that are known to play a role in these processes.

Abnormal ECM stiffness, as observed in tissue fi brosis,

clearly plays an important role in cancer progression. However,

we have only begun to decipher how different cell types respond

to changes in ECM elasticity and which receptors detect

the various types of physical force. It remains to be an important

area of research to determine whether ECM elasticity may

be restored to normal in cancer and how such a restoration may

benefi t treatment prognosis. ECM anomalies, including stiffness,

have been associated with delivery and resistance of conventional

drugs ( Egeblad et al., 2010b ). Indeed, a decrease in the

fi broblast pool and thus the ECM improves drug uptake in the

mouse ( Loeffl er et al., 2006 ; Olive et al., 2009 ). Therefore, targeting

abnormal ECM may provide yet another effective avenue

to combat the complicated illness that is cancer.

We apologize to those whose work could not be cited due to space constraints.

We thank Dr. Tim Hardingham and members of the Lu laboratory for critical

reading of the manuscript.

This work was supported by grants from Breakthrough Breast Cancer

(to P. Lu) and the National Institutes of Health (R03 HD060807 to P. Lu, R01

CA057621 and a Developmental Project from P50 CA058207 to Z. Werb,

U01 ES019458 to Z. Werb and V.M. Weaver, and R01 CA138818 to

V.M. Weaver).

Submitted: 28 February 2011

Accepted: 23 January 2012

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Tracing epithelial stem cells during development,

homeostasis, and repair

Alexandra Van Keymeulen 1 and Cédric Blanpain 1,2

1

Université Libre de Bruxelles, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), B-1070 Bruxelles, Belgium

2 WELBIO, Université Libre de Bruxelles, 1070 Bruxelles, Belgium

Epithelia ensure many critical functions of the body, including

protection against the external environment, nutrition,

respiration, and reproduction. Stem cells (SCs) located in

the various epithelia ensure the homeostasis and repair of

these tissues throughout the lifetime of the animal. Genetic

lineage tracing in mice has allowed the labeling of SCs and

their progeny. This technique has been instrumental in characterizing

the origin and heterogeneity of epithelial SCs,

their tissue location, and their differentiation potential under

physiological conditions and during tissue regeneration.

Introduction

Epithelia are sheets of cells that constitute the lining of most of

the organs of the body—such as the skin, the digestive and

respiratory tracts, and the urogenital system—and separate the

inside of the body from the outside ( Blanpain et al., 2007 ). An

epithelium can be composed of one (simple epithelium) or multiple

layers of cells (stratifi ed epithelium), and forms the majority

of the glands. A variety of specialized differentiated cells present

in these epithelia ensure the important diversity of physiological

functions they control. The skin epidermis acts as a barrier that

keeps fl uids in and protects the animal from the aggressions

of the external environment. The lungs allow the gas exchange

necessary for cellular respiration. The gut together with its associated

glands allows the absorption of water and nutrients. The

urologic system allows the evacuation of ions, water, and toxic

byproducts of metabolism. The genital tract ensures reproductive

function. Due to their vital functions, epithelia have mechanisms

to ensure their proper maintenance and functionality. Some

epithelia such as the skin or the intestine have a very high cellular

turnover to replace the cells that are continuously lost ( Barker

et al., 2010a ). Other epithelia, such as those of the airway tracts,

renew much more slowly under physiological conditions but

nevertheless can rapidly and extensively proliferate to repair

tissue upon damage or injuries ( Rock and Hogan, 2011 ). Stem

cells (SCs) located in these different adult tissues are essential

Correspondence to Cédric Blanpain: Cedric.Blanpain@ulb.ac.be

Abbreviations used in this paper: HF, hair follicle; SC, stem cell.

JCB: Review

to sustain tissue turnover and repair these different epithelia

upon injuries. SCs can renew throughout life and can differentiate

into the different cell lineages of their tissue of origin. The balance

between SC proliferation and differentiation must be precisely

controlled, as deregulation of this process may lead to tissue atrophy

and cancer formation. Carcinoma, which are tumors arising

from epithelium, are by far the most common cancers in humans

and lead to millions of death per year throughout the world.

Different assays have been developed to study the function

of epithelial SCs. Inspired by the fi eld of hematopoietic SCs,

the most common assay to assess the renewal and differentiation

of putative epithelial SCs is their transplantation into immunodefi

cient animals ( Blanpain et al., 2007 ). Although transplantation

assays are very informative about the differentiation potential

of SCs, they do not necessarily refl ect physiological conditions

because SCs are dissociated from their normal environment and

very often transplanted into a heterotopic site, such as into the

renal capsule (the fi brous layer surrounding the kidney) or the

dermis, with or without their normal underlying mesenchyme.

These assays recapitulate embryonic development or severe

regeneration conditions rather than normal tissue homeostasis.

Lineage tracing has now become the method of choice to study

the renewal and the differentiation potential of SCs in intact tissue,

as this approach avoids the many drawbacks of transplantation

experiments. In this technique, a particular cell lineage is labeled

and the fate of the labeled cells and their progeny is analyzed

over time. These experiments are usually performed in mice coexpressing

two different transgenes: a CRE recombinase expressed

under a lineage-specifi c promoter, and a reporter gene only

expressed when the CRE removes a stop cassette that precedes

the reporter gene. Upon CRE excision of the stop cassette, the

reporter transgene is permanently expressed in these cells and

all their future progeny. Two different CRE genes can be used

to perform such experiments: a constitutive CRE, which is naturally

active, and an inducible CRE, which is usually fused to a

mutated nuclear hormone receptor such as the estrogen receptor

(ER) called CREER or progesterone receptor (PR) called CREPR.

In the absence of their synthetic ligands (such as tamoxifen for the

CREER or RU486 for the CREPR), the CREER is maintained

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The Rockefeller University Press

J. Cell Biol. Vol. 197 No. 5 575–584

www.jcb.org/cgi/doi/10.1083/jcb.201201041 JCB 575

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inactive in the cytoplasm. Administration of the synthetic ligand

induces activation of the inducible CRE recombinase, which in

turn induces the expression of the reporter gene in the cells

expressing the CRE and all their subsequent progeny, allowing

the fate of labeled cells to be followed over time. The limitation

of the inducible lineage tracing experiments is the identifi cation

of a specifi c promoter that targets the SC of interest and the

mosaic expression of the reporter gene within the SC population.

The latter precludes one from drawing any fi rm conclusions

about the fate of the nonlabeled cells. In these studies, it is generally

assumed that the labeled cells are representative of the

whole SC population. Because there is an important heterogeneity

within epithelial SCs, one should always leave open the

possibility of the existence of another population of epithelial

SCs not labeled by this approach. The frequency of labeled cells

over time is a good indication of whether these cells are in equilibrium

with other SC populations; if nonlabeled SCs replace

labeled cells, the frequency of labeled cells should decrease over

time. Clonal analysis allows following the fate of single

marked cells. To perform clonal analysis, it is necessary to mark

individual cells suffi ciently distant from each other to follow

the fate of individual clones. This can be achieved by lowering

the dose of the TAM until it induces CREER activity in isolated

cells. Lineage-tracing experiments provide important information

about the cellular hierarchy that governs epithelium development

and homeostasis, and uncover the diversity of epithelial SCs,

their origin, tissue location, renewal, migration, and differentiation

potential, as well as their ability to contribute to tissue repair and

tumor initiation. Different methods to perform lineage tracing in

vertebrates and invertebrates have been recently thoroughly

reviewed elsewhere ( Buckingham and Meilhac, 2011 ; Kretzschmar

and Watt, 2012 ). In this review, we discuss the novel insights

into epithelial SCs and their lineages that have been gathered

thanks to the use of this technique in mice.

The skin epidermis

The skin epidermis is composed of the interfollicular epidermis,

which forms the skin barrier, and its various appendages such

as the hair follicle (HF), the sebaceous gland, and the sweat

gland ( Fig. 1 A ; Blanpain and Fuchs, 2006 ). During mouse embryonic

development, HFs are specifi ed through an epithelial–

mesenchymal interaction around embryonic day (E) 16. At this

stage, a group of cells that present an elongated morphology

called the placode progress to form a bud called the hair germ,

which keeps growing and differentiates into the seven concentric

cell lineages that form the mature HF ( Blanpain and Fuchs,

2006 ). Lineage tracing during embryonic epidermal development

using ShhCRE ( Levy et al., 2005 ) or Sox9CRE ( Nowak et al.,

2008 ), which are specifi cally expressed in the hair germ, demonstrated

that all HF lineages including adult HF SCs and the sebaceous

gland arise from embryonic progenitors expressing these

two markers during HF morphogenesis. Similar long-term labeling

of the HF lineages has been obtained using Lgr6CREER lineage

tracing at E17.5, which is also expressed in embryonic hair germ,

although its expression is broader and included cells of the

interfollicular epidermis ( Snippert et al., 2010a ). Embryonic

lineage tracing using ShhCRE ( Levy et al., 2005 ) and Sox9CRE

JCB • VOLUME 197 • NUMBER 5 • 2012

( Nowak et al., 2008 ) also revealed that HFs were entirely labeled

whereas the interfollicular epidermis was not labeled, demonstrating

that the interfollicular epidermis and the HF lineages

arise from two independent sources of cells that are maintained

independently in adult animals.

In addition to ensuring the barrier function and thermoregulation,

the skin epidermis is also a sensory organ that perceives all

kinds of sensory stimuli. Merkel cells are neuroendocrine cells

scattered in the epidermis that are responsible for the fi ne touch

sensation ( Maricich et al., 2009 ; Lumpkin et al., 2010 ). Merkel

cells present a number of characteristics of presynaptic neurons.

They are excitable cells that express pro-neural transcription

factors, possess many components of the presynaptic machinery,

and contact the end of sensory nerves ( Haeberle et al., 2004 ).

Due to their resemblance with neuronal cells, there has been a

long-standing debate whether Merkel cells originate from the

neural crest derivatives or from epidermal cells. Two independent

groups have recently demonstrated using lineage-tracing experiments

that Merkel cells do not originate from neural crest cells,

but rather from epidermal progenitors by a mechanism depending

on the expression of the proneural transcription factor Atoh1

in epidermal progenitors ( Morrison et al., 2009 ; Van Keymeulen

et al., 2009 ).

Transplantation experiments suggested that the permanent

portion of the HF called the bulge contains multipotent

SCs able to differentiate into all epidermal lineages including

HF, sebaceous gland, and interfollicular epidermis upon transplantation

with embryonic skin mesenchyme into immunodefi

cient mice ( Rochat et al., 1994 ; Oshima et al., 2001 ; Blanpain

et al., 2004 ; Morris et al., 2004 ; Claudinot et al., 2005 ; Jaks et al.,

2008 ). Lineage tracing of adult bulge SCs using the K15CREPR

( Morris et al., 2004 ), ShhCRE ( Levy et al., 2005 ), Sox9CRE

( Nowak et al., 2008 ), Lgr5CREER ( Jaks et al., 2008 ), and

K19CREER ( Youssef et al., 2010 ) markers confi rmed that bulge

SCs are indeed multipotent SCs responsible for the maintenance

of all HF lineages under physiological conditions ( Fig. 1, B and C ).

Clonal analysis of bulge SCs indicated that some bulge SCs

can differentiate into all HF lineages, whereas others are committed

to either the outer or the inner cell layers ( Legué et al.,

2010 ; Zhang et al., 2010 ). These results suggest that the hete rogeneity

in the differentiation potential of bulge SCs is either

related to an intrinsic heterogeneity of bulge cells containing

multipotent and unipotent SCs or through a regulation of the

lineage differentiation potential of multipotent SCs. Similarly

to the embryonic HF lineage tracing, lineage tracing of adult

bulge SCs also revealed that the HF lineages do not contribute

to the homeostasis the interfollicular epidermis under physiological

conditions ( Morris et al., 2004 ; Jaks et al., 2008 ;

Youssef et al., 2010 ), consistent with the presence of two separate

pools of SCs that separately maintain HF and interfollicular

epidermis turnover ( Fig. 1 D ). However, upon wounding,

bulge SCs are rapidly activated and actively participate in the

repair of the damaged interfollicular epidermis ( Fig. 1, E and F ;

Tumbar et al., 2004 ; Ito et al., 2005 ; Levy et al., 2007 ). These

experiments suggested that the multipotency of bulge SCs

observed in transplantation assays represents the fate of bulge

SCs in a wounding or regenerative environment. Lineage tracing

of HF matrix cells demonstrated that these cells are transient

amplifying cells that differentiate into one of the six lineages

depending on their spatial position within the matrix ( Legué

and Nicolas, 2005 ; Youssef et al., 2010 ).

JCB Highlights 2012 31

Figure 1. Lineage tracing of the skin epidermis. (A) Schematic representation of the skin epidermis and the different epidermal SCs: the interfollicular

epidermis (IFE) SCs, the isthmus SCs, and the bulge SCs . (B and C) Lineage tracing of bulge stem cells (green) using K19CREER-RosaYFP mice induced

with 10 mg tamoxifen between d 21 and 25, and analyzed 1 wk (B) or 5 wk (C) after induction. Immunostaining of CD34 (B) or integrin � 4 (C) and YFP

show the initial labeling of CD34+ bulge SC (B) and the presence of YFP cells in the newly formed hair follicle 5 wk after (C). (D) Lineage tracing of basal

cells (green) from the interfollicular epidermis using K14CREER-RosaYFP mice induced with 1 mg tamoxifen and analyzed 5 wk later. Immunostaining of

K14 (red) and YFP (green) shows the presence of a column of YFP-marked cells spanning from the basal layer (red) to the cornifi ed layer, corresponding to

a unit of interfollicular epidermis maintained by a single SC. (E and F) Migration of H2B-GFP label-retaining bulge SCs to the interfollicular epidermis after

wounding, showing the early contribution of bulge SC to the wound repair. Adapted from Tumbar et al. (2004) with permission from AAAS. (G) Lineage

tracing using Lgr6-GFP-IresCREER/Rosa-LacZ mice induced at d 20 and analyzed 1 yr later, showing the labeling of the sebaceous gland, and demonstrating

the existence of long-lived SC of the sebaceous gland. Adapted from Snippert et al. (2010a) with permission from AAAS. Bu, bulge; DP, dermal papilla;

HG, hair germ; IFE, interfollicular epidermis; SG, sebaceous gland; In, infudibulum; Ep, epidermis; w, wound. Bars, 50 μM.

Sebaceous gland SCs arise from multipotent embryonic HF

progenitors expressing Shh, Lgr5, Sox9, and Lgr6 ( Levy et al.,

2005 ; Nowak et al., 2008 ; Snippert et al., 2010a ). Within the

next few days, Blimp1-expressing progenitors are specifi ed along

Lineage tracing of epithelial cells • Van Keymeulen and Blanpain

577

32

578

the HF and give rise to the cells of the adult sebaceous gland

including sebaceous gland SCs ( Horsley et al., 2006 ). The longterm

labeling of the sebaceous gland observed after Lgr6 lineage

tracing in adult skin demonstrates the presence of resident

Lgr6-expressing sebaceous gland SCs ( Fig. 1 G ). Similar to

bulge SCs, Lgr6-expressing isthmus SCs are also recruited

toward the interfollicular epidermis upon wounding and contribute

to the repair of the damaged epidermis ( Snippert et al.,

2010a ). A recent study using new K15CREER transgenic mice

suggests that a fraction of bulge SCs migrate upward toward the

isthmus region and replenish the sebaceous gland under homeostatic

and physiopathological conditions ( Petersson et al., 2011 ).

Further studies will be required to better understand the respective

contribution of bulge versus resident Lgr6+ SCs in maintaining

the homeostasis of the sebaceous gland and defi ning

the mechanisms leading to the recruitment and the migration

of bulge SCs to this gland.

As previously mentioned, the interfollicular epidermis

is maintained by a source of SCs that is distinct from the HF

lineages during postnatal physiological conditions. Many small

units of proliferation called epidermal proliferative units (EPUs)

scattered all along the interfollicular epidermis ensure the constant

turnover of the epidermis and its proper differentiation.

Based on morphological and proliferation studies, it has been

proposed that the interfollicular epidermis is maintained by hexagonal

shaped EPUs containing one SC and � 10 transit-amplifying

cells ( Potten, 1974 , 1981 ). Retroviral fate mapping indeed demonstrated

the presence of columns of labeled cells spanning

from the basal layer to the top of the cornifi ed layer a long time

after the clonal marking ( Ghazizadeh and Taichman, 2001 ),

consistent with the presence of long-lived SCs ensuring the longterm

homeostasis of an EPU. However, genetic lineage tracing

showed that the EPU was not hexagonal in shape as suggested

by histological analysis and was sometimes bigger than the

10 basal cells expected from the model based on morphological

observations ( Ro and Rannala, 2004 , 2005 ). These data were

interpreted as the consequence of SC migration from one EPU

to another. Recently, clonal analysis studies labeling basal

epidermal cells of the tail and the ear epidermis using a nontissuespecifi

c drug inducible CREER (AhCREER) showed that the

majority of labeled cells are not maintained long-term and are

lost over time. However, the surviving clones seemed to expand

continuously, in contrast to what would be expected if these

cells were maintaining a discrete and predetermined unit of the

epidermis. Mathematical modeling of these data suggests that

the epidermis could be maintained by one type of progenitor,

without the presence of transit-amplifying cells, and these progenitors

undergo population asymmetric renewal by balancing

asymmetric and symmetric cell division in a stochastic manner

( Clayton et al., 2007 ; Doupé et al., 2010 ). Although this model

is appealing in its simplicity, it relies on the assumption that all

basal cells of the epidermis behave like the basal cells labeled

by the AhCREER. The model also does not easily explain the

ability of the tissue to respond rapidly to increased cell demand,

such as during wound healing. Importantly, these data cannot rule

out the presence of a small proportion of more quiescent SCs that

would exist in equilibrium with more committed basal cells,

JCB • VOLUME 197 • NUMBER 5 • 2012

as it has been previously suggested. New studies combining clonal

analysis with different epidermal-specifi c CREER and proliferation

kinetic data will be helpful to address these open questions.

In summary, lineage-tracing experiments in the skin epidermis

indicate the existence of multiple populations of stem cells

that ensure the maintenance of different compartments of the

epidermis and play distinct roles during homeostasis and repair.

The mammary gland

The mammary gland of the mother plays an essential role during

the early postnatal life of young mammalian offspring by providing

them nutrients, water, and electrolytes, and immune defense

until they reach the size and maturity to survive independently.

Mammary glands are epidermal appendages that are specifi ed

along the ventral epidermis around E12 during mouse embryonic

development. These glands are initially visible as placode-like

structures that progressively invade the underlying mesenchyme,

called the mammary fat pad. At puberty, the mammary gland

expands considerably to form a highly branched tubular structure

that progressively fi lls the entire fat pad. The epithelial part

of the mammary gland comprises two main cell types, the basal

myoepithelial cells and the luminal cells, which can differentiate

either into ductal cells or milk-producing alveolar cells ( Fig. 2,

A and B ). During pregnancy, the mammary gland further expands

and differentiates into milk-producing cells. After each cycle

of pregnancy, the mammary gland involutes through a massive

apoptosis of alveolar cells after which the gland returns to a

similar morphology as before pregnancy ( Watson and Khaled,

2008 ). Transplantation studies using different populations of mammary

epithelial cells isolated by fl uorescence-activated cell sorting

have demonstrated that rare single cells are able to reconstitute

an entire functional mammary gland ( Shackleton et al., 2006 ;

Stingl et al., 2006 ). These results suggest the presence of rare

multipotent mammary SCs at the top of the cellular hierarchy of

the breast epithelium ( Visvader, 2009 ). The whey acidic protein

(WAP) promoter is active in luminal cells during pregnancy

and lactation. Lineage-tracing experiments using the WAP-CRE,

which labeled luminal cells during pregnancy, identifi ed luminal

cells capable of resisting apoptosis during involution and which

clonally expand upon the succeeding pregnancy to give rise to

luminal and alveolar cells. These cells were therefore described

as alveolar progenitors and called parity-induced cells ( Wagner

et al., 2002 ). More recently, lineage tracing of the mammary

gland using inducible CRE expressed either in myoepithelial

cells (K14- or K5-expressing cells) or in luminal cells (K8- or

K18-expressing cells) demonstrated that the mammary gland

initially develops from multipotent embryonic K14-expressing

progenitors, which give rise to both myoepithelial cells and

luminal cells. However, postnatal mammary gland development

that occurred during puberty, as well as mammary gland expansion

that accompanied pregnancies, are ensured by the presence

of two types of long-lived SCs. K14/K5-expressing myoepithelial

and K8/18-expressing luminal unipotent SCs are able to

differentiate at the clonal level into myoepithelial or luminal

lineages, respectively, rather than being maintained by rare

multipotent SCs ( Fig. 2, C–H ). Decreasing the ratio of luminal

to myoepithelial SCs stimulates the luminal differentiation of

myoepithelial SCs in transplantation assay ( Van Keymeulen

et al., 2011 ). Further studies will be necessary to defi ne the

mechanisms that restrict the differentiation potential of myoepithelial

SCs under physiological conditions, and whether pathophysiological

conditions can expand the differentiation potential

of SCs in the intact mammary gland. Also, it would be interesting

to determine whether other glandular epithelia such as the

prostate or sweat glands are also maintained by the presence

of distinct classes of SCs during postnatal development and

adult homeostasis.

The gut

The gut regulates the absorption of water, electrolytes, nutrients,

and vitamins essential for the survival of the animal. The

gut can be subdivided into the esophagus, stomach, intestine,

and colon. The esophagus is a stratifi ed epithelium resembling

skin epidermis, whereas the stomach, intestine, and colon are

simple epithelia composed of only one cell layer that contains

the different cell lineages present in the different parts of the gut

( Barker et al., 2010a ).

The stomach can be subdivided into the corpus, the pylorus,

the cardia, and the fundus. In the corpus, the parietal cells

secrete the acid necessary to activate digestion, while the mucus

cells secrete a protective barrier and the enteroendocrine cells

JCB Highlights 2012 33

Figure 2. Lineage tracing of the mammary

gland. (A and B) Schematic representation of

the main epithelial cell types (myoepithelial,

luminal, and alveolar cells) of the breast during

post-natal development (A) and pregnancy

(B). (C–E) Lineage tracing of myoepithelial cells

(green) using K14rtTA/TetOCre/RosaYFP mice

induced at the onset of puberty (4 wk old) and

analyzed 1 wk (C) or 10 wk (D) later or during

lactation (E). Immunostaining of K14 (C and D)

or K5 (E) (red) and YFP (green) demonstrates

the labeling of unipotent SCs that ensure myoepithelial

lineage expansion during puberty

and pregnancy. (F–H) Lineage tracing of luminal

cells (green) using K8CREER/RosaYFP mice

induced at the onset of puberty (4 wk old) and

analyzed 1 wk (F) or 10 wk later (G), or during

lactation (H) shows the labeling of unipotent

SCs that ensure luminal lineage expansion

during puberty and pregnancy. Adapted from

Van Keymeulen et al. (2011) with permission

from Nature Publishing Group. Bars, 10 μM.

regulate gut contractility and the secretion of the various digestive

enzymes. Although the mucus cells renew quite rapidly, the

chief cells and the parietal cells responsible for acid secretion

display a slow turnover. Random marking of stomach SCs after

chemical mutagen administration demonstrated the coexistence

of long-lived multipotent and unipotent stomach SCs ( Bjerknes

and Cheng, 2002 ). In the pylorus, the vast majority of the proliferating

cells are located in the middle zone called the isthmus.

Lgr5 lineage tracing, which marked cells located at the bottom

of the gland, demonstrated that the progeny of Lgr5-expressing

cells migrate into the isthmus region, proliferate, and give rise

to all lineages of the stomach ( Barker et al., 2010b ). Recently,

Sox2 lineage tracing, which appears to mark cells distinct from

Lgr5+ cells, showed that Sox2 also marked long-lived multipotent

stomach SCs ( Arnold et al., 2011 ). Further studies will

be required to understand the relationship between Sox2+ and

Lgr5+ stomach SCs.

The small intestine ensures the absorption of nutrients.

The intestine is composed of proliferating crypts that produce

the cells giving rise to the differentiated villi. Villi are protrusions

of the epithelium in the gut lumen that massively increase

the surface area of the gut to allow for greater absorption. The

intestine is one of the most rapidly proliferating tissues in the body,

which completely self-renews in less than a week. Several cell

Lineage tracing of epithelial cells • Van Keymeulen and Blanpain

579

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580

lineages are present in the intestine: the absorptive enterocytes

are the most abundant cells, the goblet cells produce the mucus,

and the enteroendocrine cells regulate the motility of the gut and

the secretion of the digestive enzymes while the paneth cells

residing at the base of the crypt produce microbicide proteins

( Fig. 3 A ). Based on proliferation kinetics experiments, it has been

suggested that intestinal SCs reside in the crypt at position +4

with respect to the base of the crypt ( Potten et al., 1978 ).

The existence of multipotent SCs in the intestine has been

suggested by the presence of long-lived clones of marked cells

that contain several types of differentiated intestinal cells

( Bjerknes and Cheng, 1999 ). Barker et al. (2007) identifi ed

Lgr5 as a Wnt target gene in colonic cancer lines, and demonstrated

that Lgr5 is expressed at the base of the crypt in intestinal

crypt base columnar cells intercalated between paneth cells.

In contrast to the vast majority of Wnt target genes, which are

expressed throughout the crypt, including in the transit amplifying

cells, Lgr5 expression is restricted to crypt base columnar cells.

Lgr5CREER-IRES-GFP lineage tracing demonstrated that indeed

Lgr5-expressing crypt base columnar cells are rapidly cycling

multipotent SCs of the intestine giving rise to all cell lineages

of the intestine including enterocytes, goblet cells, and neuroendocrine

cells, as well as paneth cells ( Fig. 3, B and C ; Barker

et al., 2007 ).

Bmi1, a polycomb repressor, is expressed in the proximal

intestine and preferentially marks the cells located in position +4.

Cells labeled in lineage-tracing experiments using Bmi1CREER,

similarly to Lgr5+ cells, give rise to all cell lineages of the intestine

( Fig. 3, D and E ; Sangiorgi and Capecchi, 2008 ), suggesting

the existence of two distinct classes of SCs in the small intestine.

Two independent studies using CREER show that preferentially

but not exclusively labeled +4 cells (mTertCREER and Hopx-

CREER) give rise to all intestinal lineages ( Montgomery et al.,

2011 ; Takeda et al., 2011 ), confi rming the presence of multipotent

intestinal SCs in the +4 position. The faster expansion

of Lgr5 progeny in comparison to Bmi1- or mTert-derived cells

suggests that +4 intestinal SCs could be more quiescent than

Lgr5+ SCs, but yet long-lived. Interestingly, although the ablation

of Bmi1+ cells leads to the degeneration of the crypts ( Sangiorgi

and Capecchi, 2008 ), the ablation of Lgr5 cells has no effect on

intestinal homeostasis, as Bmi1+ SCs can compensate for the

loss of Lgr5 cells ( Tian et al., 2011 ). Under physiological conditions

or after the destruction of Lgr5+ cells, Bmi1 cells can replenish

the Lgr5 pool. Similarly, mTert and Hopx labeled cells

also give rise to Lgr5+ cells and conversely Lgr5 cells can give

rise to Hopx cells ( Takeda et al., 2011 ), suggesting that the Lgr5

SCs are in equilibrium with the +4 cells SCs.

Although there are around 16 Lgr5+ cells per crypt, Lgr5

lineage tracing using a multicolor reporter mouse demonstrated

that the crypt rapidly evolves from polyclonal labeling (multicolor)

to monoclonal labeling (monocolor) ( Fig. 3, F and G ).

Analysis of Lgr5 cell division revealed that they mostly divide

symmetrically giving rise to 2 Lgr5+ cells, incompatible with a

model in which homeostasis is maintained by pure asymmetric

cell division (one SC gives one transit-amplifying cell). Instead,

this supports a model in which SC divisions are symmetric at the

cellular level, but, at the level of the SC pool, the divisions seem

JCB • VOLUME 197 • NUMBER 5 • 2012

Figure 3. Lineage tracing of the intestine. (A) Schematic representation

of the intestinal epithelium lineages (enterocytes, enteroendocrine, goblet,

and paneth cells) and its SCs. (B and C) Lineage tracing of Lgr5+ SCs

(blue) using Lgr5-GFP-IresCREER/RosaLacZ mice analyzed 12 h (B) or 60 d

(B) that give rise to the differentiated cells of a villus (C). Adapted from

Barker et al. (2007) with permission from Nature Publishing Group.

(D and E) Lineage tracing of Bmi1+ SCs (green) using Bmi1CREER/Rosa

YFP mice analyzed 5 d (D) or 2 mo (E) after induction. Staining of YFP

(green) and of chromogranin A (ChGA) labeling enteroendocrine cells

(red); and lysozyme (Lys) antibody, labeling Paneth cells (red), showing

the initial labeling of cells above the paneth cells (D) that give rise to the

differentiated cells of a villus (E). Images courtesy of E. Sangiorgi and

M. Capecchi. (F and G) Lineage tracing of crypts using Ah-CREER/

RosaConfetti mice analyzed 1 wk (F) or 8 wk (G) after induction, showing

the initial multicolor labeling of the crypt and villus unit that progressively

become monoclonal (one color per crypt) over time. Adapted from Snippert

et al. (2010b) with permission from Elsevier.

asymmetric because for one SC that divides, one SC is lost, leading

to neutral drift dynamics in which SCs expand or are lost at

random ( Lopez-Garcia et al., 2010 ; Snippert et al., 2010b ).

How to reconcile the data supporting the existence of two

populations of intestinal SCs in equilibrium with each other,

with the presence of neutral drift toward monoclonality of the

intestinal crypt? One possibility is that, despite their relatively

distinct tissue localization, Lgr5 and Bmi1 are functionally equipotent

SCs competing with each other in the neutral drift proliferation

dynamics. Another possibility is that the long-term lineage

tracing in both models results from the labeling of the same multipotent

intestinal SCs, as there is a small overlap between the

cells traced with Lgr5CREER and the cells traced with Bmi1,

Hopx, mTER CREER. Further studies analyzing the rate of the

drift toward monoclonality using Lgr5+CREER and the other

+4 CREER will help to address this open question.

Although Lgr5 can also mark colonic SCs ( Barker et al.,

2007 ), the low frequency of Lgr5-marked crypts due to the

mosaic expression Lgr5CREER in the colon renders it diffi cult

JCB Highlights 2012 35

Figure 4. Lineage tracing of the airway epithelium. (A and B) Schematic representation of the airway epithelium that can be divided into trachea and

bronchi, bronchioli, and alveoli. (C–F) Lineage tracing of tracheal basal cells using K5CREER/Rosa-LacZ and analyzed 6 d (C and E), 3 wk (F), or 12 wk

(D) after TAM administration. Paraffi n sections of X-gal–stained (blue) (K5-labeled cells and their progeny) and anti-acetylated tubulin (brown, cilia), showing

the initial labeling of basal cells (C) that give rise to luminal cells during postnatal development (D). (E and F) Lineage tracing of tracheal basal cells

using K5CREER/Rosa-YFP and analyzed 3 and 15 wk after TAM administration showing the initial labeling of basal cells (green) that give rise to Clara

cells (red). Adapted from Rock et al. (2009) with permission from Proc. Natl. Acad. Sci. USA . (G–I) Lineage tracing of the Clara cells in the bronchioli

using Scgb1a1CREER/RosaYFP mice induced at E18.5 and analyzed 2 d (G), 3 wk (H), or 1 yr (I) after induction. Immunostaining of GFP (green), pro-

SPC (AEC2) (red), and Scgb1a1 (Clara cells) (blue) shows the long-term renewal of Clara cells and the expansion of ciliated cells over time. Arrowheads

represent lineage-labeled AEC2 cells. Arrows represent lineage-labeled putative BASCs. Inset in H shows labeled ciliated cells (arrowheads), but no neuroendocrine

cells (red, arrow). The stable frequency of lineage-labeled AEC2 cells over time (1–3%) suggests that BASC cells do not contribute to alveoli

expansion during postnatal growth. Adapted from Rawlins et al. (2009) with permission from Elsevier. AEC1, alveolar type I; AEC2, alveolar type II; BADJ,

bronchioalveolar duct junction; BASC, bronchioalveolar stem cell. Bars: (C and D) 20 μM; (E and F) 25 μM; (G) 50 μM.

to ascertain whether there is one or more colonic SCs contributing

to the homeostasis and the regenerative potential of the

colonic epithelium.

The airway system

The airway system is compartmentalized along the proximal–

distal axis into three anatomically distinct regions: the trachea

and bronchi, the bronchioles, and the alveoli. The cellular composition

of the lung epithelium varies in the different regions, as

well as the cellular hierarchy that regulates the maintenance and

repair of these epithelia ( Rock and Hogan, 2011 ). The trachea

and bronchi are pseudostratifi ed epithelia containing ciliated

cells, secretory cells (Clara cells and goblet cells), and basal

cells ( � 30% of tracheal epithelial cells; Fig. 4 A ). The function

of this fi rst part of the airway tract is to allow the circulation of

the air but also to protect the lung epithelium by absorbing dust

particles and microbes that are constantly inhaled. Through

movement of the cilia, these cells chase the dirty mucus out of

the respiratory tract. Rare neuroendocrine cells are also dispersed

Lineage tracing of epithelial cells • Van Keymeulen and Blanpain

581

36

582

in the luminal layer of the trachea, which regulate the contraction

of the respiratory tracts as well as the secretion of the mucus

( Rock and Hogan, 2011 ).

In contrast to the constant and rapid turnover occurring in

the intestine or the skin epidermis, the airway system presents

a very low renewal under steady-state conditions. For example,

lineage tracing using Foxj1-CREER, which labeled ciliated

cells, supports the view that ciliated cells are postmitotic with

a half time of several months under homeostatic conditions

( Rawlins et al., 2007 ). There is increasing evidence that basal

cells function as multipotent SCs in the trachea and bronchi,

able to self-renew and differentiate into both Clara cells and

ciliated cells under steady-state conditions and after injury.

The fi rst evidence that basal cells contain multipotent SCs

came from lineage-tracing experiments using the K14-CREER

to label basal cells in the trachea and bronchi during naphthaleneinduced

epithelium regeneration, demonstrating the massive

contribution of basal cells during trachea and bronchi regeneration

( Hong et al., 2004a , b ). However, under physiological

conditions most mouse basal cells express K5, whereas only

a subset of basal cells expresses K14, which is up-regulated in

the basal cell population upon injury. These studies did not

assess the contribution of the basal cells under steady-state

conditions. Rock et al. (2009) used a K5-CREER to label basal

cells of the upper airway tract postnatally under physiological

conditions and demonstrated that basal cells contain selfrenewing

multipotent SCs, giving rise to Clara and ciliated

cells during postnatal growth, adult homeostasis, and epithelial

repair ( Fig. 4, C–F ). Lineage tracing using Scgb1a1 (also called

Secretoglobin 1a1 or CC10) CREER mice, which specifi cally

marked Clara cells in the trachea and the main bronchi, demonstrated

that some of these cells can undergo several rounds of

replication giving rise to Clara and ciliated cells. However,

most of them are progressively lost and replaced over time by

cells that are not marked by Scgb1a1CREER ( Rawlins et al.,

2009 ), suggesting that Clara cells in the trachea and the main

bronchi do not contain SCs and behave as a transit-amplifying

cell population.

Bronchioles lack basal cells but are surrounded instead

by myofi broblast cells ( Fig. 4 B ). They are mainly composed

of ciliated and secretory cells, and also contain clusters of neuroendocrine

cells. Lineage tracing of Clara cells with Scgb1a1-

CREER showed that Clara cells from bronchioles self-renew

over a long period of time and give rise to ciliated cells, during

postnatal growth, homeostasis, and repair of the bronchiolar

epithelium. This is consistent with the presence of bipotent

Scgb1a1-expressing SCs, which ensure the homeostasis of the

bronchioles ( Fig. 4, G–I ; Rawlins et al., 2009 ).

Alveoli are the sites of exchange between the inhaled air

and the gas of the blood. To maximize the surface of exchange

between the blood and the air, the bronchioles end in multiple

sacs, called alveoli, which are encased by a dense capillary network.

The alveoli contain two major cell types: the alveolar

type 1 cells, which are squamous cells that comprise the major

surface of the lung and express different ion transporters critical

for fl uidity of the mucus produced, whereas alveolar type 2 cells

are cuboidal cells that secrete the surfactant protein C (SPC)

JCB • VOLUME 197 • NUMBER 5 • 2012

essential to maintain the alveoli open. Proliferation kinetic

experiments suggested that the alveolar type 1 cells are terminally

differentiated cells that do not divide, neither under physiological

conditions nor during tissue regeneration. On the other hand, type

2 cells divide during homeostasis and repair, and were assumed

to contain SCs of the alveoli ( Adamson and Bowden, 1975 ),

although no lineage-tracing experiment has formally demo n strated

this hypothesis. Cells expressing both Scgb1a1 (the Clara marker)

and pro-SPC (a marker of cuboidal alveolar type 2 cells) have

been described at the bronchioalveolar junction. These cells do

not die upon injury, become proliferatively active, and exhibit

SC properties in culture, and therefore were called bronchioalveolar

SCs ( Kim et al., 2005 ). Labeling of bronchiolar cells,

including the bronchioalveolar SCs, with the Scgb1a1-CREER

demonstrated that these cells do not contribute to the alveoli

during postnatal growth and after hyperoxia injury but rather

contribute to the bronchiolar regeneration ( Rawlins et al., 2009 ).

However, a new study showed that cells labeled with the

Scgb1a1-CREER contribute extensively to the alveoli regeneration

after bleomycin-induced lung injury, suggesting that the

contribution of Clara cells to lung regeneration can vary depending

on the type of injury ( Rock et al., 2011 ). Consistent with the

role of Scgb1a1+ cells during lung repair after bleomycininduced

injury, lineage tracing of alveolar type 2 cells using

SPC-CREER demonstrated that SPC-derived cells are replaced

by SPC-negative progenitors during lung repair after

bleomycin inhalation ( Chapman et al., 2011 ). K14-CREER lineage

tracing suggested that K14/K5-positive cells appear in

the bronchioles a few days post-infection of H1N1 virus and

give rise to migrating K5-positive clusters of cells at the sites of

interbronchiolar lung damage ( Kumar et al., 2011 ). However,

the origin of these K5-positive cells remains unclear. Do they arise

from Sgbd1a1 cells that begin to express K5/K14 upon injury

or do they come from already K5-positive cells from the main

bronchia? What is their long-term contribution to alveoli lineage?

Further work will be needed to clarify these open questions.

Perspectives

Lineage-tracing experiments in different epithelia reveal the

coexistence of several types of SCs in each epithelium. Epithelia

such as the epidermis and the intestine contain rapidly cycling

SCs, dividing asymmetrically at the level of the population but

symmetrically at the SC level, which balanced renewal and

differentiation stochastically. In addition, these tissues present a

slower cycling population of cells that represent a reserve pool

of SCs in case of sudden need. More studies will be required to

precisely determine how the equilibrium between these two pools

of SCs is achieved and what the molecular mechanisms are that

allow the stochastic choice between renewal and differentiation.

Another theme that emerges from these lineage-tracing

studies is the existence of both multipotent and unipotent SCs

maintaining the diversity of cell lineages found in these epithelia

during postnatal life. Further studies will be necessary to better

understand when the switch from multipotency to unipotency

occurs during morphogenesis, how the differentiation of these

epithelial SCs is controlled, and what the relative importance of

intrinsic versus extrinsic determinants is in regulating their fate.

New lineage-tracing studies will be required to identify

new types of SCs in the different epithelial tissues. For example,

how are the esophagus, pancreas, bladder, prostate, and ovary

developed, maintained, and repaired? Do these tissues contain

multipotent SCs or different classes of unipotent SCs? The identifi

cation of the different SCs, transit-amplifying cells, and differentiated

cells in these different epithelia will be instrumental

to uncover the cell lineages at the origin of the different epithelial

cancers ( Visvader, 2011 ). What is the respective importance

of oncogenic mutations versus the cellular origin in dictating

the tumor phenotypes? Also, the identifi cation of cellular origin

of the different epithelial cancers will allow one to defi ne more

precisely the transcriptional and genetic changes accompanying

tumor initiation and to understand the molecular mechanisms

that shape the fate of tumor-initiating cells. Cancer SCs have

been identifi ed in several human and mouse cancers based on

their ability to reform primary tumors after transplantation into

immunodefi cient mice ( Lobo et al., 2007 ). Further studies will

be required to demonstrate the existence of cancer SCs during

in vivo tumor growth in intact tissues using lineage-tracing and

clonal analysis.

We thank Brigid Hogan and Ben Simons for insightful discussions. We apologize

to those whose work could not be cited due to space constraints.

C. Blanpain and A. Van Keymeulen are Chercheur Qualifi é of the

FRS/FNRS. C. Blanpain is an investigator of Welbio. This work was supported

by the FNRS, TELEVIE, and the program d’excellence CIBLES of the Wallonia

Region; research grants from the Fondation Contre le Cancer, the ULB foundation,

and the fond Gaston Ithier; a starting grant of the European Research Council

(ERC); and the EMBO Young Investigator Program.

Submitted: 9 January 2012

Accepted: 27 April 2012

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