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Proliferating Cells-Brdu

+ Retinoic acid

DAPI / BrdU / Nestin / Merge

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C ell C ycle Times

M any human cell types have cell cycle times of ~1 dayM itosis is usually the shortest phase (usually ~1 hour)S phase is usually 1/3 to 2/3 of cell cycle (can't be too short)

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F igure 14.4. Determination of cellular DNA content A population ocells is labeled with a luorescent dye that binds N A. The cells are then

passed through a low cytometer, which measures the luorescence

intensity o individual cells . The data are plotted as cell number versusluorescence intensity, which is proportional to N A content . The

distribution shows two peaks, corresponding to cells with N A contentso 2 n and 4 n; these cells are in the G 1 and G 2/M phases o the cycle,respectively . C ells in S phase have N A contents between 2 n and 4 n andare distributed between these two peaks .

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A

D

B

CG0-G1- 80.0%G2- M -9 %S-11%

G0-G1- 80.5%G2- M -9.5%

S-20.9%

G0-G1- 63.7%G2- M -15.4%

S-20.9%

G0-G1- 80.4%G2- M -8.4%

S-11.2%

C ell C ycle A nalysis By Flow C ytometer

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Restriction point or G 1 /S TransitionRegulated by protein phosphorylation,protein degradation, and inhibitory proteins

G 2 /M Transition - Entry into M phaseRegulated by protein phosphorylation

Anaphase onset - Exit from M phaseRegulated by protein degradation

R egulatory Mechanisms

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S. cerevisiae (budding yeast)

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Cell Cycle

G1: Gap 1, Va r i ti

: DNA s t sisG2: Gap 2M: Mit sis

Nutrients

Matingactors

C ell size

- R egulation by Extra cellular signals S .cerevisiae .

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C ell C ycle R egulation

K ey cell cycle transitions : 1. Restriction point (in late G 1; regulates S phase onset)2. G 2 - M transition (in late G 2, regulates M phase onset)

3. M etaphase-anaphase transition (regulates exit from M )

1

2 3

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Cell cycle checkpoints

C ell cycle checkpoints prevent entry in to the next phase o the cellcycle until the events o the preceding phase have been completed .These checkpoints sense unreplicated or damaged N A and coordinateurther cell cycle progression with the completion o N A replication

or repair .For example, the checkpoint in G2 prevents the initiation o mitosisuntil N A replication is completed .This G2 checkpoint senses unreplicated N A , generates a signal thatlead to cell cycle arrest .

peration o the G2 checkpoint there ore prevents the initiation o M phase be ore completion o S phase, so cell remain in G2 until thegenome has been completely replicated .

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A rrest at the G1 checkpoint allows repair o thedamage to take place be ore the cell enters S

phase .The S phase checkpoint provides continualmonitoring o the integrity o N A to ensure thatdamaged N A is repaired be ore it is replicated .A complex o sensor proteins binds to damaged

NA and activates the A TM and A TR proteinkinases . These inturn phosphorylate and activatethe checkpoint 2 and checkpoint 1 kinases leadingto cell cycle arrest .

Cell cycle checkpoints

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R ole o 53 in G1 arrest

In mammalian cells arrest at the G1 check point ismediated by additional protein known as 53 which is

phsophorylated by A TM and C hk 2 .

hsophoyrlation stabilizes p53, p53 is a trascriptionalactor and an increased expression lead to induction otarget genes that induces, cell cycle arrest .Loss o p53 unction is usually ound in human cancersdue to mutation .

Spindle check point monitors the alignment ochromosomes on the mitotic spindle to ensure that acomplete set o chromosomes are distributed accurately tothe daughter cells .

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R estriction o N

A R

eplicationNA replication is restricted to once per cell cycle

by M C M protein that bind to origins o replicationtogether with R C (origin replication complex)

protein and are required or the initiation o N A replication .MC M proteins are only able to bind to N A inG1, allowing N A replication to initiate in S

phase. nce initiation has occurred, the M C M proteins

are displaced, so that replication cannot initiateagain until a ter mitosis .

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R ole o 53 in G1 arrestinduced by N A amage

strand break

p53 p21

-tyr15

cdk2

cyclin E

p21 = CK I class (cyclin dependent kinase inhibitors) N-terminal o p21 orms complex with cyclin / cdk - inhibit

kinase

X-rays

ATM

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D NA D amage - C ell C ycle A rrest

damage dependent checkpoints

CELL No .

D NA content D NA content

asynchronousX-ray treatedG1/S block G2/M block (6-9 hours)

G1 - S - G2

wild-type

loss o G1/S in p53 de icientcells

G1 - S - G2

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C ell C ycle C heckpointsCheckpoints are quality control steps in the cell cyclethat determine if the cell is prepared to progress to thenext stage of the cell cycle and ensure that one cell cyclephase has been completed before moving to the next

Questions asked at checkpoints :Environment good? (G 1)Growth factors present? (G 1)DNA damage? (G 1, S, G 2)DNA replicated? (S)DNA replication errors? (G 2-M )M itotic spindle properly formed? (G 2-M )Chromosomes attached to spindle? (anaphase transition)

Checkpoints are enforced by regulation of CD K

activities

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MPF: M - as e P r ti Fa c t r

Iden tifica ti n f MPF

1971- Yoshio M . and C lement M . , D ennis S . R obert E . ,

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Mitotic C D K (M F) A ctivation

1. C D K protein level changes little during the cell cycle2. M itotic cyclin levels rise in G 2 and reach a critical threshold3. A ctive C D K /cyclin complex yields a peak of kinase activity4. C D K complex phosphorylates proteins and drives G 2 to M

5. Cyclin is degraded to inactivate C D K and permit exit from M

Cyclin protein level

Cyclin-dependent kinase (C D K ) protein level

Cyclin-Cdk kinase activity

G2 M

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Second A pproach understanding C ell C ycle R egulation

Schizo saccharomyces pombe- aul Nurse characterized thismutants

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FRO

M YEA

ST TO

MA

N: cdk sconservation o sequence across evolutioncdk1 = G2/M transitionission yeast (S pombe) = cdc2

budding yeast (S cerevisiae) = cdc28man = cdc2 homolog; p34cdk2 = G1/S transitionission yeast (S pombe) = cdc2 (S orm)

budding yeast (S cerevisiae) = cdc28man = cdk2; p33 - S A LR E moti

R egulators o C ell C ycle rogression

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MPF a c tivit y is ependen t u pon Cycl in

Acc umu la ti n and deg r ada ti n f c clins

Third line o investigation- In sea Urchin embryo

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MPF: M -ph as e P r omo ting Fa c to r

MPF is c mpo s ed of t o key s ubun its :

dc2 and yclin . dc2 is t he pr ote in tha t en coded by gene s

hich a r e r equ ir ed f or pa ss age thr ough TART a s e ll a s f or en tr y into m itosis.

yclin is a r egu la tor y s ubun it r equ ir ed f or ca ta lytic a ctivity of the dc2 pr ote in kina s e .

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MPF: M -ph as e P r omo ting Fa c to r

S tr uctur e o f MPF

Molecular C haracterization o M F rom rog egg-James Maller

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MPF a c tivit y is dependen t u pon Cycl in

The cyclins e r e iden tified a s pr ote ins t ha t

a cc umu la te thr oughou t inte r pha s e and a r e r ap idly deg r aded towa r d the end of mitosis.It is s ugge st ed tha t they migh t f un ction to indu ce mitosis, with the ir pe riod ic

a cc umu la tion and de str uction con tr olling en tr y and ex it fr om M pha s e .

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MPF r eg u la tion

C dc2 f or ms c omp lexe s with cyclin du ring S and G2 .C dc2 is t hen pho s pho r yla ted on thr eon ine -161 , wh ich is r equ ir ed f or C dc2 a ctivity, a swe ll a s on tyr osine -15 (and thr eon ine -14 in ve rteb r a te ce lls), wh ich inh ibits C dc2

a ctivity. D epho s pho r yla tion of Thr 14 and Tyr 15 a ctiva te s MPF a t the G2 to M tr an sition .MPF a ctivity is t hen te r mina ted towa r d the end of m itosis by pr oteo lytic deg r ada tion of cyclin .

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MPF r eg u la tion

Demon str a tion of r egu la tion of MPF

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Fa m ilie s o f Cycl in s a nd Cycl in-ependen t Ki n as e s

B oth C dc2 and cyclin B a r e membe rs of la r ge

f am ilie s of r e la ted pr ote ins, with diff e r en tmembe rs of the s e f am ilie s c on tr olling pr og r e ssi on thr ough distinct pha s e s of the ce ll cycle .The ce ll cycle s of highe r eu ka r yote s a r e con tr olled

no t on ly by mu ltiple cyclins, bu t a lso by mu ltiple C dc2-r e la ted pr ote in kina s e s. T he s e C dc2-r e la ted kina s e s a r e known a s Cdk' s (f or c yclin-d ependen tk ina s e s). C dc2 is a lso known a s C dk1, with othe rs

be ing de signa ted C dk2 C dk .

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H uman have multiple cyclins and multiple C D K sSpecific cyclin-C D K complexes are active during specific

cell cycle stages (C D K complexes are not active in G 0)

C D K s and C ell C ycle

D iagram showsCD K activitylevels during thehuman cell cycle

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Fa m ilie s o f Cycl in s a nd Cycl in-ependen t Ki n as e s

In yea st, pa ss age thr ough S

TART is c on tr olled by C dc2 in a ss ocia tion with G1 cyclins ( C ln1 , C ln2 ,and C ln3 ). C omp lexe s of

C dc2 with distinct B -type cyclins ( C lb 's) t hen r egu la te pr og r e ssi on thr ough S pha s e and

en tr y into mitosis.

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Fa m ilie s o f Cycl in s a nd Cycl in-ependen t Ki n as e s

In an ima l ce lls, pr og r e ssi on thr ough the G1 r e stricti on po int is c on tr olled by comp lexe s of C dk4 and C dk6 with D-type cyclins. C dk2/cyclin Ecomp lexe s f un ction la te r in G1 and a r e r equ ir ed f or the G1 to S tr an sition .C dk2/cyclin A comp lexe s a r e

then r equ ir ed f or pr og r e ssi on thr ough S pha s e , and C dc2/cyclin B comp lexe sdrive the G2 to M tr an sition .

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T emperature-sensitive mutations

These are mutations that give rise to mutant proteins thatexhibit an increased (or decreased) sensitivity to temperature .

For example, a change rom one amino acid to another at acritical location in the protein can result in a protein that

is unstable at 37oC , the nonpermissive temperature . Thus i that protein is essential or normal cell growth,

then the cell will not grow . R educing the temperature to 30 oC ,the permissive temperature, may allow the cell to grow .

Cd c mutants ail to progress through the cell cycle;wee mutants are d e ective in proteins that preventcells rom d ivid ing prematurely (check points) .

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20-12: R ecessive an d d ominant c d c2 mutants have opposite phenotypes . R ecessive c d c2 - mutants cannot enter mitosis at the non-permissivetemperature an d thus appear as elongate d cells with a single nucleus . The d ominant c d c2 D mutant enters mitosis prematurely, an d is thus wee .

The wee cell

Loss o the kinase activity

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F o ur molec u la r mech a n is m s th a tr eg u la te c tiviti e s o f Cdk' s

Me chan ism s of C dk r egu la tion

CAK -cd k-activatingkinase

Cd c25 amily- phosphatases

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R egulation By

C D K I hibit ( CK I )

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C D K Inhibitors ( CK Is)

CK Is bind to and inactivate C D K /cyclin complexes

p27Inhibitory

D omain

Cyclin A

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3. Regulate cell cycle : Rb gene: that inhibits the cell cycle in the G1 phase

decrease cell proli eration

.

INK-4 gene: that pro d uces 16 that inhibitscd k4/cyclin D action ( to phosphorylate R b gene toinactivate its action)

P53: that pro d uces 21 that has the same action o16 in inhibiting the action o c d k4/cyclin D

Function o Tumour Suppressor

gene

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C ell cycle regulation of Rb and E2 F In its un d erphosphorylate d orm,R b bin d s to members o the E2F amily, repressing transcription o E2F-regulate d genes . hosphorylation o R b by Cd k4, 6/cyclin D complexesresults in its d issociation rom E2F in late G 1. E2F then stimulatesexpression o its target genes, which enco d e proteins require d or cellcycle progression .

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Regulate cell cycle

Rb Rb

PP16

C ell C ycle Blocked C ell C ycle Proceeds

R b inactive

Cd k4/cyclin D

G1

M G2

SS

G1

M G2

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E2F re ers to a amily o at least ived istinct transcriptional actors . It isnoteworthy that only three members o E2F

interact withR b

.The other two E2Fs mayinstea d be regulate d by other R b-relate d

proteins . owever the interaction o E2F byR b represent a key mechanism responsibleor controlling cell cycle progression .

E2F: Transcriptional Factor

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Inh ibit o r o f Cell Cycle P r og r e ss ion

A va rie ty of signa ls a ct t o inh ibit ce ll cycle pr og r e ssi on .The e ff e cts of inh ibitor y signa ls a r e a lso med ia ted by r egu la tors of the ce ll cycle ma chine r y, fr equen tly via the indu ction of C dk inh ibitors.

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Inh ibit o r o f Cell Cycle P r og r e ss ion

DNA damage r e s ults i n the e leva tion of intr a ce llula r leve ls of p53 , wh ich a ctiva te str an scri ption of the gene en cod ing the C dkinh ibitor p21 . In add ition to inh ibiting ce llcycle pr og r e ssi on by bind ing to C dk/c yclin

comp lexe s, p21 may dir e ctly inh ibit DNAs ynthe sis by inte r a cting with P C NA (a s ubun it of DNA po lyme r a s e d ).

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Inh ibit o r o f Cell Cycle P r og r e ss ion

Indu ction of p21 by DNA damage

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Inh ibit o r o f Cell Cycle P r og r e ss ion

A c omp lex of che ckpo int pr ote ins r e cogn ize sun r ep lica ted or damaged DNA and a ctiva te sthe pr ote in kina s e C hk1, wh ich pho s pho r yla te sand inh ibits t he C dc25 pr ote in pho s pha ta s e .Inh ibition of C dc25 pr even ts depho s pho r yla tion

and a ctiva tion of C

dc2.

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Inh ibit o r o f Cell Cycle P r og r e ss ion

C on tr ol of the G2 che ckpo int

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Tumor Suppressor p53

p53 gene encodes a 53 k D a protein

p53 protein functions as a transcription factor

p53 responds to DNA damage - guardian of the genomep53 can arrest cell cycle progression or promote apoptosis

A bout 50% of human tumors contain mutations in p53 gene

M ost point mutants map to DNA -binding regions in p53(inability to bind DNA causes loss of tumor suppressor activity)

Some DNA tumor virus oncoproteins bind to and inactivate p53

53 F ti

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M dm2 binds to p53,causing its degradationDNA damage triggersphosphorylation of p53(stable and active)

p53 induces expressionof p21, a C D K inhibitor

p21 arrests the cellcycle, which allowsrepair of DNA damage

p53 can also trigger apoptosis rather thancell cycle arrest

p53 Function

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Stages in C arcinogenesis

p16 - a CD inhi itor

p53 - t mor s ppressor

M F an d progression to Meta phase

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T argets of MP F M F in d uces multiple nuclear an d cytoplasmic changesat the onset o M phase, both by activating other protein kinases an d by

phosphorylating proteins such as con d ensins an d the nuclear lamins .

M F an d progression to Meta phase

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Dissolution of t e nuclear lamina The nuclear lamina consists o ameshwork o lamin ilaments . A t mitosis, Cd c2 an d other protein kinases

phosphorylate the lamins, causing the ilaments to d issociate into reelamin d imers .

Meta hase Spin d le

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Meta hase Spin d le

M d l f l f i b AP C l d

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Model for control of entry into anap ase b y AP C -regulateddegradation of t e co esin link b etweensister c romatids.

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Fig . 3-29:a) The ubiquitin-me d iate d

proteolytic pathway .

Ub is a small pepti d ethat is activate d and

then attached

to atarget protein, or to itselon a protein .

b) A mo d el o the proteasome .

It recognizes ubiquitinate d proteins an d d egra d es themto constituent pepti d es .

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MCB Fig. 13-9

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Cytokinesis in animal cells

D uring cytokinesis in animal cells the cell membrane squeezes together aroun d the mi dd le o the cell .The cytoplasm orms into two cells me d iate d by contractile ring o actin an d

myosin two ilaments that orms beneath the plasma membrane.

Each d aughter cell gets about 1/2 o the organelles .

C ytokinesis in plant cellsBecause the plant cell has a cell wall unlike the animal cell, it cant squeezetogether like the cell membrane can .

Instead a structure calle d the cell plate orms across the mi dd le o the cell an d the cell gra d ually splits into two cells .Then two new cell wall orms .

O verview of eukaryotic cell cycle regulation

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y y g

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C ell C ycle A nalysis By Flow C ytometer

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A

D

B

C0-G1- 80.0%G2- M -9 %S-11%

G0-G1- 80.5%G2- M -9.5%

S-20.9%

G0-G1- 63.7%G2- M -15.4%

S-20.9%

G0-G1- 80.4%G2- M -8.4%

S-11.2%

C ell C ycle A nalysis By Flow C ytometer

C heckpoint an d C ore Layers

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p y

Checkpoint layer - collects information about environment,cell (e.g, DNA ) damage, and completion of cellular processes

Core layer - C D K and A PC activities drive progression of cell

cycle (thick arrows) and are regulated by checkpoints

C ytokine sis in animal cells

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A cknowle d gement

I thank Mr . Nawaz S .S. or all the helptowar d s this presentation

I thank D r . Manjula or the aca d emic helptowar d s this lecture .

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Thanking you