BMB - control of cell cycle

why study the cell cycle

cell division is coordinated for correct development

conserved in all eukaryotes

must be coordinated for homeostasis

can be perturbed in rare genetic diseases

cancer therapies inhibit cell cycle

cell cycle principles

system of biochemical oscillators to allow complete synthesis and segregation of DNA

series of all or nothing switches

unidirectional

governed by checkpoints that make it responsive to cues

cyclins

transcribed and degraded

co factor for CDKs

each cyclin causes synthesis of following cyclin

catalyse their own destruction

CDKs

phosphorylate targets to change their activity

initiate physical events and regulatory events

G1/S CDK-cyclin

CDK4/6 and cyclin D

G1/S and S CDK-cyclin

CDK2 and cyclin E

S CDK-cyclin

CDK2 and cyclin A

M CDK-cyclin

CDK1 and cyclin B

transcription and degradation of cyclins

transcription controlled by E2Fs - 1, 2 and 3 are activators and 4 and 5 are repressors

degraded by APC/C

action of CDKs

phosphorylate and activate aurora, polo and greatwall kinases

these are weaker substrates of APC/C and so are degraded later

function of aurora kinase

phosphorylates histone H3 causing condensation

function of polo kinase

phosphorylates cohesin causing sister chromatid separation

function of greatwall kinase

phosphorylates ENSA causing mitotic entry

INK CKIs

bind cyclin D CDK4/6 to prevent S phase entry

p16 and p21

degradation of CKIs

constantly ubiquitinated by SCF E3 ligases in G1/S transition

F box is co activator

substrate targeting determined by phosphorylation

PP1

inactive when phosphorylated by CDK1-cyclinB

PP1 slowly autophosphorylates to activate

counters CDK activity e.g. dephosphorylates greatwall

greatwall kinase effect on mitotic entry

1. greatwall kinase activated by CDK1-cyclinB

2. phosphorylates ENSA

3. ENSA-P inhibits PP2A allowing mitotic entry

4. PP2A slowly dephosphorylates to become active

inactive greatwall = less ENSA phosphorylation and more active PP2A - mitotic gatekeeper

checkpoints

G1/S - START - RP = checking if enough resources

SAC = checking if chromosomes are ready

G2/M = checking DNA replication

why use embryonic cell cycle as model

rapid division without growth

no checkpoints

large numbers of cells

divisions happen quickly

uncouple growth from division

unregulated

experiment - identification of MPF in xenopus

take cytoplasm from egg and inject into an oocyte = maturation without progesterone

oocyte normally arrested after S phase of meiosis

xenopus model system

easy to inject

large cell size

experiment - oscillation of cyclins

add 35S methionine to embryonic extracts

western blot shows oscillations

cell free extracts

can directly add or remove protein

manipulate protein levels

addition of mutants

removal using antibodies

retain cytosolic organisation

isolation of MPF

affinity chromatography

suc1 isolated from yeast pull down CDK1-cyclin B on Sepharose beads

conformation of cyclin B transcription

1. RNase treat extracts

2. block RNase and add cyclin mRNA

3. extract goes through 3 cell cycles

shows cyclin B is regulatory factor of MPF

evidence of cyclin B D box

first 90 residues

loss = no degradation

addition of excess D box = no degradation of wt cyclin B

experiment - APC/C role

block poly Ub addition with methyl-ub

cyclin B destruction blocked

degradation is delayed

APC/C co factors

cdc20 = degrades cyclin B and securin, active in anaphase, upregulated by M phase cyclinB-CDK1

cdh1 = degrades cyclin A, polo, aurora, geminin and cdc20, active in M/G1, inhibited by S phase cyclinA/E and CDK2

ensuring DNA replication only happens once

G1 = loading of helicase, replication licensing

S = activation of helicase and recruitment of polymerase, geminin inhibits replication licensing in S, G2 and M

geminin

prevents re replication

inhibits Cdt1 which normally loads helicase in G2, S and M

geminin degraded in G1 by APC-cdh1

use of S.pombe

growth in G2 makes phenotype clear - elongated rod

haploid - recessive mutations seen

process of forward genetic screen

1. devise screen for cells that are unable to go through a process

2. mutagenesis

3. identify cells with phenotype change that means they can’t go through process

4. identify genes through sequencing or complementation

5. confirm with targeted mutagenesis

advantage of forward genetic screens

non presumptive

limitations of forward genetic screens

hard to observe phenotypes

not good for polygeny

some genes may have multiple functions

reductionist

s.pombe mutants identified

Cdc2 and Cdc25 = large cells = blocked entry into mitosis

wee1 = small cells = progresses into M phase too early

cdc25 rescues wee1

cdc mutants cant pass checkpoints = CDKs

wee mutants don’t have product that inhibit START passage

suppressor screen

start with mutant

try to rescue phenotype

identify a second mutation that suppresses the first

types of suppression

allele specific - specific physical interaction e.g. Suc1 to Cdc2

high copy number suppression - high amounts of another protein can overcome phenotype caused by another protein

CDK1 regulation by wee1 and cdc25

wee1 kinase phosphorylates on tyr15 = inactive

cdc25 dephosphorylates = active

CDK1 activates cdc25 and inactivates wee1 = positive feedback

autophosphorylation on thr167 essential for activity

DNA damage response

Chk checkpoint kinases activated

act on cdc25 to inhibit and block CDK1 activity

S.cerivisae screen

can see chromosomes under microscope

screen for defects in cohesion - GFP on Scc1 = should see 1 dot if centromeres are coherent

cohesin structure

ring of smc2 and smc1 closed together by scc1

separation of centromeres

APC/C degrades securin in M phase = separase active

separase cleaves Scc1

evidence of centromere separation from yeast

mutate separase = no separation

separase identified as cysteine protease

mutate Scc1 cleavage site = no separation

engineer Scc1 cleavage with TEV protease recognition = can restore separation

use of mammalian cells as model system

flat and large = good for imaging

fluorescent proteins used

synchronisation of cells with double thymidine block and nocodazole

fucci system

G1 = red - Cdt1 replication licensing factor degraded in S

early S = yellow - overlap

S/G2/M = green - geminin replication licensing inhibitor

experiment - dose response of mammary gland to topoisomerase inhibitor

low conc = normal cell cycle

med conc = nuclear mis segregation

high = endoreplication

spindle arrangement

MTs alternate between phases of growth and shrinakge to find kinetochores

tubulin added near kinetochores and removed at other end

astral MTs position spindle

role of spindle machinery

centromeres have gamma tubulin ring complexes - MT nucleation sites

kinesins and dyenins walk along MTs exerting force to induce separation

aurora B kinase localises at centromeres to readout tension

spindle assembly checkpoint - unattached kinetochore

MAD1 binds unattached kinetochores and changes conformation of MAD2 so it can bind other co inhibitors

BUBR1, BUB3, cdc20 and MAD2 inhibit APC/C

APC/C can’t degrade securin

experiment - G1/S

timelapse record cells and determine time of birth

serum starvation allows determining age

starve in G1 or 0 = long delay to enter mitosis

starve in G2/M/S = cells enter M phase normally

shows that point after G1 there is a checkpoint for nutrients

models for cell size homeostasis

control of time between divisions

cell size dependent cell cycle progression

cell size dependent growth rate adjustment

Rb function

Rb represses E2Fs

mitogens activate cyclinD/CDK4 to phosphorylate Rb which dissociates from E2F

increase Rb = longer G1 phase

cell size experiments

measure volume by fluorescence exclusion

measure mass by quantitative phase microscopy

inverse correlation between initial mass and cell cycle phase