Regulation of the Eukaryotic cell cycle Flashcards
(20 cards)
Cell cycle and exp evidence overview
Cell cycle mech conserved across euk. Coordinated for homeostasis, incl for wound healing, epithelial renewal. Cell cycle dysregulation can -> rare genetic diseases (e.g. microencephaly- most cells in the body are in brain- cell division disorders impact brain most); fertility loss (older oocytes have less sister chromatid cohesion). Most cancer therapies target cell cycle (e.g., paclitaxel-> cytokinesis failure).
Exp evidence for cell cycle: mechs evolutionarily conserved- use best exp system for each process (ease of manipulation key, gather evidence from combo of models)- cell free extracts (Xenopus, sea urchin), genetic screens (budding+ fission yeast); mammalian cells- synchronisation+ microscopy.
Underlying rationale of cell cycle and phases of mitosis
Underlying rationale: cell cycle= biochemical oscillator to allow complete synth+ segregation/DNA, a series of all/nothing switches. Growth+ division are coordinated- they’re unidirectional+ repeat, alternating between S (DNA rep, growth) + M phase (mitosis). Cycle is governed by checkpoints responding to internal+ env cues.
Phases of mitosis are visually distinct- late interphase (centrosome duplicates) prophase (chr condense, centrosomes separate), prometaphase (nuclear env breakdown, kinetochores attach MTs), metaphase (chr align @centre of spindle under tension), anaphase (sisters synchronously separate), telophase (2 nuclear envelopes reassemble), Cytokinesis (cytoplasm division).
Switches, CDKs and cyclins
Switches can be made irreversible by: creation+ degradation/important molecules, transcriptional ctrl; UQ-mediated degradation, +ve feedback loops (enzyme destroying own inhibitor)
CDKs regulate physical (H1 Pi to help chr condensation, Pi of MT-assoc proteins) and regulatory events (cdk/cyclin B stimulates own activation, triggers proteolysis system to degrade cyclin B) events in mitosis
Cyclins are transcribed+ degraded to ctrl the cycle, activate CDKs which Pi target proteins. G1 phase- CDK4+6, cyclins D1,2,3; G1/S phases CDK2+ cyclin E (cyclin A just S phase); M CDK1+ cyclin B. Cyclin transcription is tightly ctrled b specific TFs- E2Fs 1,2,3 are activators+ E2F4+5 are transcriptional repressors. Cyclin degradation is ctrled via APC/C (anaphase promoting complex/cyclosome- large 13 subunit complex That UQs proteins) and proteasome (recruited to protein by addition of UQ chain)
Event ordering by protein complexes
Events are ordered by CDK/cyclin complexes Pi’ing another tier of kinases w/ consensus Pi site S/T-P-X-K/R found across proteins Aurora, Polo+ Greatwall in Drosophila. Substrate ordering is based on kinase/substrate availability, substrate affinity+ targeting. Aurora, Polo, etc weaker substrates/ APC, degraded later than cyclin B
CDK/cyclin B-> nuclear lamins-> nuc env breakdown
Aurora-> H3-> chr condensation
Polo->cohesin-> sister chromatid separation
Greatwall->ENSA-> mitotic entry
CDK/cyclin activity control by cyclin-dependent kinase inhibitors and degradation pathway. Checkpoints and quality control
CDK/cyclin activities also ctrled by cyclin-dependent kinase inhibitors (CKIs): CIP/KIP (p27,p21,etc) proteins bind range/ cyclin-CDK complexes, stopping cell primarily @ G1/S boundary, sometimes @ later checkpoints. Direct transcriptional target of tumour suppressor p53, which is elevated in response to DNA damage, increasing p21-> arrest at G1. INK fam/proteins bind cyclin D/CDK4,6, preventing activation inhibiting G1-S transition.
CKIs are UQ’d, targeted for degradation by SCF E3 ligases in G1-s transition: Constant activity during cell cycle, substrate targeting determined by Pi status od F-box binding. SCF-F-box complex UQs CKIs
Ordered degradation+ de-Pi turns mitosis off:
1) CDK substrates Pi’d by cyclin-CDK complexes+ de-Pi’d by PP2A-B55, while CDK-cyclin complexes+ their de-Pi’d substrates are subject to proteasome mediated degradation
2) CDK1/cyclinB de-Pi’s, inactivates PP1, which has slow rate of autodePi. When CDK1/cyclinB leels drop, PP1 slowly reactivates (timer mech)
3) CDF1/cyclinB Pi’s, activates Greatwall. Phospho-ENSA inhibits PP2A-B55- slowly reversible by autode-Pi. CDK1/cyclinB degraded, PP1 inactivates Greatwall. ENSA dePi’d. PP2A-B55 activated-> dePi substrate. System is tunable by ENSA level
Checkpoints provide quality ctrl: G2/M checkpoint for correct replication; spindle assembly check for chromosome readiness; START/restriction point/ G1/S checkpoint for if enough resources available for division
Evidence for ordered transitions between cell cycle stages: clams/sea urchins/frogs and Xenopus oocyte, S-35 methionine
Embryonic cell cycles of clams/sea urchins/frogs- rapid unregulated division w/out growth- no G stage/ checkpoints means ctrl+ underlying oscillator can be studied. This is because: enough nutrients for growth until offspring can feed; enormous #s counter predation; larva form ASAP; growth uncoupled from division
Xenopus oocyte maturation+ fertilization process: oocyte growth+ arrest after meiotic S phase; progesterone induces oocyte maturation, egg arrested in metaphase of meiosis II; fertilization triggers completion of meiosis II, chromosomes segregate into polar bodies that form. This implies the existence of a catalytic maturation promoting factor that, when injected into oocyte (from cytosol of mature egg) repeatedly induces meiosis.
Rapid dev/ sea urchin to free swimming larva similar to frog, can only experiment in summer due to lifecycles
Xenopus often best for studies because can year-round experiments, they’re easy to inject/large, population synchronous in divisions, extracts sustain several cycles to support biochemical elucidation
Adding 35S-methionine/ embryonic extracts-> oscillatory rise+ fall/ cyclin, implicating it as a cycle co-activator.
Evidence for ordered transitions between cell cycle stages: experimental protocols, cell free extracts, MPF
Cell free extracts can recapitulate a process in a way that can be directly visualised, allow immediate +/- extra recombinant proteins (no membrane)/ specific removal of proteins w/ antibody, + manipulate extract composition while maintaining cytosolic composition. Esp good for timelapse studies (can film rhythmic divisions)
Gentle crushing spin of frog egg retains activity of cell cycle machinery, Ca2+ activated, extracts cycle.
MPF (maturation promoting factor) activity peaks on oocyte maturation, meiosis II activation+ in mature oocyte, then at each division- it can be isolated by affinity chromatography p13Suc1 (yeast multicopy suppressor of cdc2ts), confirming its identity as CDK1/cyclinB. Further evidence of cyclin B involvement= confirmation of its transcription- RNAse treat extracts (remove mRNA), block RNAse+ add back just cyclin RNA- capable of cycling extract through 9 cycles, proving cycling dependent on cyclin B as regulatory factor of MPF- cyclin degradation explains loss of MPF.
Evidence for ordered transitions between cell cycle stages: cyclin B trunchation, UQ steps and E3 ligase
Cyclin B trunchated @ 1st 90 aas (containing “destruction box”) persists, H1 kinase activity constant. This shows that degradation of cyclin critical for MPF activity decline- embryos stay in mitosis. Further proof:
UQ chains are important for degradation: cyclin degeneration in clam extracts (model for UQ-mediated degradation) shows methylUQ can’t form polyUQ chains-> cyclins A/B degradation delayed until methylUQ outcompeted by UQ.
Ubiquitination happens in 3 steps: ATP/UQ-> ADP, adding UQ to E1; E1/UQ transfers UQ-> E2, E2/UQ ubiquitinates the target protein/ adds to UQ tail. E3 ligase (APC/C) selects the substrate
E3 ligase= multiprotein complex, can purify from Xenopus/ clam egg. 1 wave of APC/C triggers anaphase, 2nd wave persists late mitosis-> through G1. It has 2 activators: Cdc20-> degradation/ cyclin B+ securing @ anaphase exit; Cdh1-> degrade cyclin A, polo-/aurora-like kinases, geminin+ cdc20 on mitosis exit. Protein degradation complements transcriptional ctrl to regulate protein levels. CDK/cyclins turn AOC isoforms on/off for switch-like regulation in protein levels+ therefore cell cycle phases.
DNA replication only happens once per cell cycle due to…
Geminin= rep licencing inhibitor, prevents re-rep/ DNA in same cycle-> genomic stability. ID’d by fractionation of frog egg extracts for inhibitors of DNA rep. Targeted for degradation in early G1 for licensing, accumulates again in S/G2+M. Overall result: G1 phase- loading of DNA helicase (replication licencing) ensures DNA unwound for replication, then S phase activation/ helicase, polymerase recruitment.
Cyclin A also inhibits loading helicase so loading only in G1 when APCCDH1 active. In S phage, cyclin A accumulates+ doesn’t allow further helicase loading as helicase activated+ DNAPols recruited.
Evidence for error checking mechanisms: S pombe mutants (forward genetic screens and mutant summary)
S pombe- most growth in G2, making phenotypes clear, helping study the G2/M transition.
Forward genetic screens helpful because: non presumptive; prior knowledge not needed. Steps: screen for cells unable to undergo process of interest (too small/large) by mutagenesis by chemical/insertional methods+ IDing cells w/ relevant phenotype; ID gene by seq/complementation; confirm gene by targeted inactivation
Haploids/mating: pombe usually haploid (recessive genes show up)- ts mutants helpful for finding phenotypes. Genes cloned by complementation. Fission yeast key mutants (phenotypes @ end of mitosis, compared to wt):
Cdc2 (ts) Elongated, can’t divide
Cdc25 (ts) Elongated, can’t divide
Wee1 (ts) Small
Cdc25+wee1 Normal (rescue)
5 cdc25 copies Small
5 cdc2 copies Normal (rescue)
Cdc mutations block mitosis, define genes w/ products required to pass through checkpoint. Mutants stop @ various points. Wee mutations allow cells to enter mitosis prematurely- mutants deficient in product inhibiting passage through size checkpoint.
S pombe checkpoint mutant suppressor screens, complementation and G2/M checkpoint/Cdc25 phosphatase
Suppressor screens ID how mutant proteins/genes interact w/ mutant, attempting to rescue it. Start w/ mutant, attempt rescue; further mutagenesis/ overexp from high copy number plasmid library. Allele specific suppression= 1 allele suppressed by another specific allele, indicates specific physical interaction between them. High copy # suppression= high amounts/ another protein can overcome phenotype (rescue growth @ restrictive temperature)
Complementation demonstrates S pombe Cdc2= S cerevisiae Cdc28= human CDK1
G2/M checkpoint responds to incomplete replication/ DNA damage/double strand breaks, blocks mitosis via checkpoint kinases (Chk1+2 activate wee1 kinase, inhibit cdc25 phospatase). Wee1 kinase inhibits CDK1/cyclin B (Pi CDK1 Tyr15), so mutations mean CDK1/cyclin B active in smaller cells, allowing division. Cdc25 phosphatase de-Pi’s Tyr15, activates CDK1/cyclin B+ triggers G2-M transition. Auto-Pi of CDK1 on Thr167 essential for activity. Thr14 Pi inhibitory. CDK1 is activated by +ve feedback loop inactivating wee1+ activating cdc25, causing switch-like behaviour.
S cerevisiae studies for sister chromatid separation, separase and its role
S cerevisiae used to study how cells ensure sister chromatid separation: screen for defects in chromatid cohesion/ separation, viewing by FISH (/light microscopy, timelapses possible), e.g., Scc1-. ID Scc1 as coding for the protein closing the gap in cohesion rings (visible by EM).
Separase= Cys protease that cleaves Scc1. Securin binding+ Pi inhibit separase protease activity until needed. Securin+ cyclin B are degraded by APC/C-cdc20 in mitosis, allowing separase activity-> disrupt Scc1, releasing sister chromatids
Evidence for separase role: separase mutants fail to separate sister chromatids; separase show to be Cys protease+ cleavage sites in Scc1 ID’d- cleavage site mutants also don’t separate sister chromatids. Re-engineering another cleavage site for TEV protease restores separation-> almost normal.
Consequences of aneuploidy
1) E.g. Trisomy 21-> Down’s- non-fatal bc chr 21 gene poor (trisomy 1 not observed bc gene rich, lethal)
2) Changing gene dosage increases housekeeping burden, can trigger stress response
3) G1 delay, can give cancer replication/fitness advantage (90% cancer cells aneuploid- lack of error check)
Spatial and size elements in cycle control (cultured mammalian cells, FUCCI system)
Cultured mammalian cells: flat, large cells (imaging, incl timelapse); exp fluorescent proteins; cell synching; double thymidine block (inhibits DNA synth to sync @ S phase, treat again to sync @ next S); nocodazole inhibits mitotic spindle to synch M phase
FUCCI system (fluorescence ubiquitination-based cell cycle indicator) allows tracking of live-cell cycle progression. Based on 2 fluorescent fusion proteins:
* mCherry-hCdt1 Human Cdt1 (replication licensing factor) fused to RFP. Cdt1 selectively degraded in S phase+ beyond, dominates in G1
* mVenus-hGeminin- human geminin (replication inhibitor) fused to GFP, selectively degraded in late M/G1, dominates in S/G2/early M
Both geminin+ Cdt1 have specific “destruction boxes” (degrons) recognised by E3 UQ ligases @ different points in cell cycle leading to overall effect: G1 primarily red fluor, Early S overlap appears yellow (red degrading while green increases); S/G2/M primarily green, M-G1 transition returns to red.
Response of Fucci2-exp mammary gland cells to DNA topoisomerase inhibitor= G2 arrest, nuclear mis-segregation, endoreplication in NMuMG cells treated w/ diff concs/ chemotherapy drug etoposide
3 stages of chromosome segregation. Mitotic spindle role
3 stages ensure accurate chromosome segregation: Cohesin binding in S phase; spindle formation+ chromosome binding under tension in M, securin/ cyclin B degradation+ separase activity in M phase.
Mitotic spindle: aligns sister chromosome. Kinetochore attached MTs to chromosome during prometaphase (ablation of 1 kinetochore sufficient to block anaphase), centromere assembles kinetochore, centrosome (enriched w/ gamma-tubulin ring complexes acting as MT nucleation sites)organises MTs, astral MTs emanate away from spindle to cell cortex to position spindle in cell centre. MTs continuously alternate between growth+ shrinkage phases to ‘search and capture’ kinetochores. Tubulin subunits added near kinetochores, removed @ - ends near poles. Flux contributes to tension across sister chromatids, accurate alignment+ segregation. Astral MT+ cortical polarity proteins position spindle in right orientation. Mitotic spindle E-intensive- careful balance of ATP use needed for accuracy of chromosome segregation to avoid aneuploidy. Kinesins+ dyneins walk along MTs, exert force for chromatid separation. Aurora B kinase localises @ centromeres/ kinetochores, reads out tension signal.
Mitotic checkpoint complex and spindle assembly checkpoint
Mitotic checkpoint complex: assembles w/ APC/C coactivator Cdc20. Cdc20 interacts MAD2 (made by conf change causing it to dissociate from MAD1 attached to kinetochore). Cdc20/MAD2 complex recruits BUBR1+BUB3, whole complex joins APC/C-> inactive complex. Complex active when APC/C-Cdc20 dissociate from the others. MAD2 kinetochore attachments gradually disappear in anaphase as MTs bind. Injection of anti-MAD2-> cell enters anaphase before proper chromosome alignment (which can -> improper segregation). Cyclin B localises to spindle MTs, condensed chromatin, centrosomes, cytoplasm.
Spindle assembly checkpoint: check for unattached kinetochores. Formation of mitotic checkpoint complex MAD2, BUBR1m Cdc20 inhibits APC/C, then once all correct, free Cdc20 coactivates APC/C, which degrades securin+ cyclin B.
Checkpoints in cancer, restriction point
Checkpoints in cancer not all equal: functional mitotic checkpoint prevents catastrophic chromosome mis-segregation+ is rarely compromised in cancer. The DNA damage checkpoint is often compromised. Replication stress checkpoint genes rarely mutated as they help tolerate high replication stress. The decision to leave the cell cycle is overcome in transformed cells.
Restriction point= G1/S checkpoint: using 3T3 cells (mouse embryonic fibroblasts) timelapse recording of cells to determine time of birth, then serum-free media (arrests post-mitotic cells in G1) for 0.5, 1, +up to 8 hrs+ determine age @ time of serum starvation. Determine time taken to enter mitosis- young cells (G0/G1, <4hrs) have long delay/ don’t start while older (G2/M/S) cells enter mitosis normally- this establishes a G1/S checkpoint for whether sufficient nutrients are available to continue through the cycle.
Growth is coupled to cell cycle timing. ..There are 3 models for cell size homeostasis…
Growth is coupled to cell cycle timing: prok cells often appear to grow exponentially, evidenced by volume of bacterial cells increasing approx. exponentially from birth to division, due to many biosynthetic reactions scaling in proportion to existing cell mass. However, Euk cells sometimes described by linear increases in cell size (single cell mass/ volume in yeast/mammalian cells linear, due to rate-limiting steps+ regulatory checkpoints dominating over growth).
3 models for cell size homeostasis: “timer model” (ctrl time between divisions), “sizer model” (size-dependent cell cycle progression), “adder model” (cell size dependent growth rate adjustment).
Cyclin regulation, Rb mutations, mitogens and MTOR. How cell size is measured experimentally
Cyclin exp regulated by system of TFs (E2Fs, see above) + a network of co-repressors (RB family)- RB (retinoblastoma, 1st tumour suppressor gene) inhibits E2Fs 1,2,3 (cyclin activators), p107+p130 repress E2Fs 4,5 (repressors).
Rb mutations-> 99% retinoblastoma (rare inherited disease) + spontaneous mutations are common in many cancers. Rb targeted by some inhibitory viral proteins (adenovirus, HPV)-> no cyclin B TF inhibition.
Mitogens activate cyclin-D/CDK4 to Pi Rb proteins, releasing them from E2F, allowing cyclin E transcription. CDK4/6 inhibitors have potential as anti-cancer drugs.
Cell size regulates G1 duration: MTOR helps sense nutrients-> activate ribosome neogenesis, promote cell growth. Potential involvement of quorum sensing (bacteria)/ similar processes in other organisms- gradient in conc across rod-shaped cell can help determine its size. Different scaling of +ve&-ve cell cycle regulators ctrl G1 length- as cell size increases, amount of inhibitors (RB) stays constant while amount of activators (cycD, E2F, etc) increases.
Cell size measurements indicate ctrl throughout cell cycle as well as G1- Inverse correlation between initial mass+ cell cycle phase duration in both G1 and non-G1 in HeLa. Adder-like correlation between birth+ division; size coordination between mass-dependent cell cycle regulation+ growth rate modulation. Indications of ctrl of anabolic+ proteostasis machinery as well as G1 duration
Cell size measured by volume fluorescence exclusion (vol)+ computationally enhanced qualitative phase microscopy (mass).
Graph of cyclin/CDK expression at different cell cycle stages
