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Syncitial Divisions in Drosophila 

embryo. From Bill Sullivan UCSC 

267A: Cell Cycle I 

Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center, 

Department of Biological Chemistry 

Office: 549 BSRB 

email: tlane@mednet.ucla.edu 

These notes are posted on the www page! 

Dr. Lane LECTURE #1 1 

http://bio.research.ucsc.edu/people/sullivan/images.html

Goals 

Features of Cell Cycles and some basic methodology 

Source of materials: 

Lectures 1-5 follow the course produced by Dr. Herschman (2006-07) 

Lecture 6 - T.B.D.

The two “musts” of cell division: 

1. Cells must replicate their DNA 

2. Cells must segregate the DNA to the 

progeny cells in mitosis 

Syncitial Divisions in Drosophila 

embryo. From Bill Sullivan UCSC 

http://bio.research.ucsc.edu/people/sullivan/images.html

How do we study the cell cycle? 

Mitotic cells can be identified under a microscope. 

Mitotic cells 

INTERPHASE 

Microscopic studies carried out early in 

the 20th century described mitotic 

cells and identified a period of “supposed” 

quiescence called interphase. 

How do we identify other features of 

cycling cells?

interphase cell Mitotic cell 

Root tips from 

Vica faba 

Faba salad 

What was happening in 1950? 

Cell cycle: 

Model: 

? 

Question: When is nuclear material synthesized? 

http://whatdidyoueat.typepad.com/what_did_you_eat/2006/05/braised_fava_be.html 

Howard and Pelc (1951) [Exptl Cell Res 51: 2: 178]

Alma Howard’s experiment: 

interphase cell 

Mitotic cell 

Expt 1] Label with 32 P, 

autoradiograph 

immediately 

Results Expt #1: 

i. no incorporation into M cells, 

ii. incorporation into some interphase cells. 

Interpretation: 

Interphase contains a period of DNA 

synthesis that is completed before M. 

Model: 




Mitotic cell 


add colchicine, wait 2 

hrs. 


• For 2-4 hours, no mitotic cells are labeled! 

i. After six hours, some mitotic cells are labeled; 


There exists a period of several hours prior to M in which 

no DNA synthesis occurs. Cells labeled for >6 hours 

passed through a period of DNA synthesis, after which 

a “Gap” period where no DNA synthesis occurred. 

Model: 

= 2-4hr gap before labeled 

Mitotic cells appear. 




Mitotic cell 


add colchicine, wait 

longer (6-8 hrs). 


i. When [32P] was added for 1hr, then removed for 

6-8 hours, some of the cells entering mitosis 

were not labeled. 


This implies there is a period between mitosis and the 

beginning of DNA synthesis when no DNA synthesis 

occurs, another Gap. 

Model: 

http://www.nature.com/nature/journal/v426/n6968/full/426759a.html 


Today, we label cells with dNTP precursors like [3H]-thymidine (TdR) 

or analogs like BrdU. 

30 Minute Pulse with 3 H-TdR, 

Autoradiograph Immediately 

Schematic of cultured fibroblasts 

grown on TC plastic.

Today, we label cells with dNTP precursors like [3H]-thymidine. 

Result #1 



Some 

interphase 

cells labeled 

( ), some 

not ( ).

Today, we label cells with dNTP precursors like [3H]-thymidine. 

Result #2 



No 

labeled 

mitoses

Can we get cell cycle information from such an experiment? 

Expt 4: Prepare a group of identical plates containing your favorite 

cell type. Each plate contains randomly growing cells 

distributed (randomly) around the cell cycle. 

PLAN: Add tritiated thymidine for 30 minutes 

Wash out the labeled thymidine 

Add colchicine, to prevent microtubule polymerization 

At intervals (e.g., one hour) fix a plate and count 

Collect the following data 

1. The number of mitotic cells 

2. The number of radiolabeled mitotic cells


Expt 4: 

Number 

of 

mitotic 

cells 

Data: 1. The number of mitotic cells 

2. The number of radiolabeled mitotic cells 

time 

RESULT: there is a steady increase in 

mitotic cells following colchicine treatment, 

finally reaching a plateau as all cells pass 

into M.

As time passes, 

some mitoses 

become labeled 

30 Minute Pulse with 3 H-TdR, add Colchicine, 

Autoradiograph at later times.


Expt 4: Data: 1. The number of mitotic cells 


Number 


mitotic 

cells 

time 

Number of 

3 H-thymidine 

labeled 

mitotic cells 

RESULT: there is a delay in the appearance 

of labeled mitotic cells following colchicine 

treatment of 3 H-Thy pulse-labeled cells

Expt 4: Data: 1. The number of mitotic cells 


Number 


mitotic 

cells 

time 


3 H-thymidine 

labeled 

mitotic cells 

RESULT: there is a delay in the appearance 

of labeled mitotic cells following colchicine 

treatment of 3 H-Thy pulse-labeled cells



M we are 

looking at 

DNA Synthesis Gap 

M M 

Lag equals a Gap 

following S, before cells 

enter M.

A further incr. in 

# of unlabeled 

mitotic cells, 

until a steady 

state is reached. 

30 Minute Pulse with 3 H-TdR, add Colchicine, 

Autoradiograph at later times.

Expt 4: 

Number 


mitotic 

cells 



time 


3 H-thymidine 

labeled 

mitotic cells 

RESULT: a plateau of labeled Mitotic 

cells is reached. The plateau is reached 

prior to the plateau in total M cells.



Gap DNA Synthesis Gap 

M M 

The time of increased 

labeling equals S



Gap DNA Synthesis Gap 

M M 

S 

G 1 

The additional time until 

the total number of M cell 

plateaus represents the GAP 

prior to S. 

G 2

Expt 4: 

Number 


mitotic 

cells 



G 2 

S 

G 1 

time 


3 H-thymidine 

labeled 

mitotic cells 

RESULT: A time line for each stage of 

the cell cycle can be obtained.

Current representations of the “Cell Cycle” 

G 2 

M 

G o 

S 

G 1 

Features: 

i. S has discrete beginning 

and end. 

ii. S is separated from M by 

Gaps. 

iii. S, G2 and M periods of the 

cycle are are of fairly consistent 

lengths. 

iv. Differences in cycle time are 

usually due to alterations in G1. 

v. In non-dividing tissues most. 

cells are frozen in G1, or "Go".

Current representations of the “Cell Cycle” 

G 2 

M 

S 

G 1 

Features: 

vi. The “Generation Time” is 

the length of a complete 

cell cycle M---M. 

GT = G 1 + S + G 2 + M 

GT = Generation time

DETERMINING THE CELL CYCLE PARAMETERS 

G1, S, G2 and M: 

G 2 

M 

S 

The classic method is known as labeled mitoses. 

1. count labeled mitoses after a 

pulse with a DNA label (eg [3H] TdR) 

2. M can be determined as the percent 

of cells that appear as mitotic figures 

in a randomly growing population. 

G 1 

S-phase cells are 

labeled after a short 

“pulse” with tritiated 

thymidine



G 2 

M 

S 

The classic method is known as labeled mitoses. 

1. count labeled mitoses after a 

pulse with a DNA label (eg [3H] TdR) 

2. M can be determined as the percent 

of cells that appear as mitotic figures 

in a randomly growing population. 

G 1 

S-phase cells are 

labeled after a short 

“pulse” with tritiated 

thymidine



Method of Labeled Mitoses: 


labeled 

mitoses 

G 2 

S 

Time 

M = GT / (%M) 

(%M is determined microscopically) 

G 2 + M + G 1



Method of micro-fluorimetry 

(FMF) or FACS: 

Cells stained with DNA 

intercalating fluorescent dye: 

Examples: 

DAPI 

Hoescht 33258 

Propidium Iodide 

Topro3 

Excitation 

(lasers) 

Emission 

Intensity is proportional to amount 

of die bound: 

G1 = 2n 

S = 2-4n 

G2 = 4n 

Capillary 

Flow



Method of micro-fluorimetry (FMF), Flow cytometry, FACS: 

Cell cycle analysis by flow cytometry requires: 

A single cell suspension of the cells of interest. 

Cells must be permeable (detergent, fixation) 

DNA in cells can be stained with a fluorescent dye 

DNA probes like Propidium Iodide are STOICHIOMETRIC 

allow quantitative assessment of DNA content 

Basic protocol - fix, wash twice, stain with DNA-binding dye (remove RNA)




(FMF) or FACS:





# of Events 

G1(2n) 

S 

G2 (4n) 

Increase in Fluorescence Intensity 

Derek Davies, Cancer Research UK






G1(2n) 

S 

G2 (4n) 






(FMF) or flow cytometry: G1 S G2 

When coupled with FACS, 

cells can be isolated for 

biochemical analysis. 


M 



MAJOR features of the cell cycle are “conserved” in 

all eukaryotes: 

Conserved Features: 

i. Replication of DNA (S) 

ii. Chromosome 

segregation (M) 

G 2 

M 

S 

G 1 

iii. Highly accurate 

duplication of DNA 

(3.2pg, 7ft of W/C DNA 

in humans)

A large number of eukaryotic systems are used to 

study various aspects of cell cycle machinery: 

Systems: 

i. Yeast 

ii. Many invertebrate embryos 

(clams, sea urchins) 

iii. Frog oocytes 

iv. Mammalian cells in culture. 

Fly (W. Sullivan www) 

Xenopus (J. Smith www) 

Fibroblasts (Thulberg et al) 

Yeast (Kerry Bloom www) 

We will draw heavily on mammalian, Xenopus, 

and yeast experiments in this class. (Biochemisty and genetics)

We can “synchronize” populations of cells at various 

points in the cell cycle. 

G 2 

M 

S 

G 1 

R=Restriction Point 

i. Starving cells leads to G1 arrest (R) 

(serum, isoleucine, or PO4 ) 

ii. Several agents can be used to 

block S (HU, nucleotide analogues, 

aphidicolin, TdR).




(FMF) or flow cytometry: 

S phase block 

Agents like 

HydroxyUrea (HU), 

nucleotide analogues, 

aphidicolin 

fluorodeoxyuridine (FdU) 

methotrexate uridine (MU) 

TdR (more in a minute) 

G2 

G2 

G2 

0 hrs 

24 hrs 





(FMF) or flow cytometry: 

G2 phase block 

Agents like 

DHAQ 

ICRF (chelating agent) 

Polo Kinase inhibitors 

G2 

G2 

G2 

0 hrs 

24 hrs 




Single Thymidine Block: 

General Method 

Treat cells w/ .25mM Thymidine for 24 hours 

(or a timed greater than G2+M+G1) 

Release by washing 2x, then proceeding as required for experiment. 

M 

DNA synthesis 

inhibitor 

M 

Cell Number 

Release and count 

cells at intervals after release 

Position of Block 

Time 

Benefits: Provides moderately pure S cells. 

Very gentle, tolerated by many types of cells.



Double Thymidine Block: 

M 

Cell Number 

Position of Block 


Treat cells w/ .25mM Thymidine for a timed greater than G2+M+G1 

wash 2x and culture for a time greater than S, less than G2+ M+ G1. 

Treat cells w/ .25mM Thymidine for a timed greater than G2+M+G1 

Release by washing 2x, then proceeding as required for experiment. 

M 

Cells released following 

1st block 

2nd round 

Thymidine 

Release and count 

cells at intervals after release 

Time 

Benefits: Provides very pure S cells. 

Very gentle, tolerated by many types of cells.



Mitotic Selection (aka Mitotic Shake-off) : 


Gently wash off loose cells and collect them. 

Increase yield by treating with Nocodazole 

(µ-tubule inhibitor) 

Theory 

Adherent tissue culture cells adhere to the plate, 

but “round up” as they enter M. This in an opportunity to isolate 

them. 

Benefits: Provides relatively pure M/G1 cells. 

Very gentle, no biochemical manipulation. 

Mitotic cells



Comparison of non-toxic methods : 

Single TdR block G 1/S and S 

Double TdR block G 1/S 

Mitotic selection M/G 1



Centrifugal Elutriation : 


Single cell suspension. 

Special centrifuge configuration that allows collection of 

samples during the run!. 

Theory 

Cells increase their size as 

they go through the cycle 

M< G1 < S < G2. 

Benifits: 

Very large numbers/volumes 

of cells. 

Can enrich for many stages. 

Excellent for blood/yeast 

Yeast Cell cycle mutants: J Bahler (Sanger Center)



Centrifugal Elutriation : 

Confirmation of separtation by FMF 

Cell 

number 

G 1 

S 

G 2 

Control early mid-E mid-L late 

fluorescence intensity

CHALLENGES IN CELL CYCLE: DNA SYNTHESIS 

cpm 

G 

1 

Questions: 

RATE 

AMOUNT 

S 

G 

2 

What initiates DNA synthesis? 

Is the signal positive or negative? 

How is synthesis restricted? 

How is chromatin regulated during 

the cycle? 

Is DNA synthesized in a random 

or ordered fashion? 

Is DNA synthesized early in one 

cycle always synthesized early? 

M 

2X 

1X

QUESTIONS CONCERNING DNA SYNTHESIS: 

Is DNA Synthesized sequentially or randomly? 

In 1960s there was cytochemical evidence suggest that "early replicating" 

and "late replicating" DNAs exists. (X-chromosome, others) 

Mueller and Kajiwara asked “Is DNA that is synthesized early in one cell cycle 

synthesized early the next cell cycle? 

PLAN: Synchronize HeLa cells with a double-thymidine block at G1/S (S = 6hr). 

Release from block, add [ 3 H]-TdR for 3 hours (ie, during the first half of S). 

Then chase with "cold" thymidine media. 

Grow the [ 3 H]-TdR labeled cells for a few generations. (loose synchrony). 

Then resynchronize with a second double-TdR block at G1/S. 

Release in the presence of BUdR for 3 hours. 

Isolate DNA, shear the DNA, and centrifuge on CsCl gradient. 

Follow radioactivity and density. 

OUTCOMES: If early replicating DNA is resynthesized early, then [ 3 H] will 

always go with the BUdR density label. 

If DNA synthesis is random, then [ 3 H] will be present in all fractions 

Mueller and Kajiwara, BBA 114: 108 (1966)



Synchronize 

cells, release 

into 3 H-TdR for 

three hours 

3 H TdR BUdR 

Let grow several 

generations, 

then resynchronize, 

label with BUdR 

for three hours 




RESULT: [ 3 H] AND BUdR co eluted. 

3 H TdR 

HL LL 

Density 

Optical 

density 

CONCLUSION: 

DNA SYNTHESIZED EARLY IN ONE 

CYCLE IS ALSO SYNTHESIZED EARLY 

IN THE NEXT CYCLE

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