Cell growth + division ALL Flashcards
1
Q
Stages of cell cycle
A
- G1, S, G2, M
- Microscopy = useful for describing events
- Yeast = genetic / mechanistic info
2
Q
Experiment
Order of cell cycle
A
- Fused G1 + S w/ virus induced fusion
- G1 immediately replicated DNA
- S phase has factor that triggered DNA replication
- Fused G2 + S
- G2 x replicate its DNA
- Fused G1, S or G2 to M2 → entry into mitosis so M has dominant activity
3
Q
Experiment
Control of cell cycle
A
- Temp sensitive mutant of CDC (grow at ↓ not ↑ temp)
- Find what complements cdc mutant
4
Q
Experiment
Rate limiting step of cell cycle
A
- Fission yeast cells add to end
- Mutants that divide at smaller size
- I mutant = wee1, entered S phase but M prematurely (x 2nd growth)
5
Q
Activation of CDKs
A
- Need cyclins + to be phosphorylation
- Phosph on T161 by CAK (activates)
- Phosph on T15/Y14 by wee1 (inhibits), cdc25 reverses
- CyclinB-CDK1 assembled + immediately inhibited in G2
6
Q
Cdc25/Wee1 regulation
A
- CyclinB-CDK1 ↑ during G2
- Threshold in late G2, phosph. Cdc25 (activates) + phosph. (inhibits)
- When cell exits = reversed
- Rapid change
7
Q
Cyclin degradation
A
- Mutagenesis showed 1st 90 aa of cyclinB has D-box, recognised by ubiquitin
- Ubiquitin recognised by proteasome
8
Q
APC/C
A
- In M, APC/C active via phosphorylation by Cdc20
- Immediately inhib. by MAD/Bub until chromosomes aligned
- Then cyclin-B destroyed
9
Q
Tubulin
A
- Building block of spindle
- and 0 end
- If meets chromosome, becomes stabilised
10
Q
Centrosome
A
- Formed around centrioles, 2 centrioles liked per centrosome
- Centrosome organises - ends of microtubule at opposite end of cell
- Overlapping microtubules captured by Eg5
- Dynein = at - pole
11
Q
Kinesin + dynein
A
- Force generating ATPases
- Move chromosomes
12
Q
Kinetochore
A
- Kinetochore proteins
- CENPA + CENPC form template on DNA, NDC80 + KNL1 sit on top
- When attached, microtubule pulls back, generates force
- TIRF microscopy, NDC80 + CENP-T track + end of microtubule
13
Q
Spindle attachment geometry
A
- Correct = amphitelic
- Incorrect = monotonic, systelic + merotelic
- Need way of sensing
14
Q
Aurora kinases
A
- Adopt similar active conformation to cyclin bound to Cdk?
- Phosph to stabilise AS and have nearly identical consensus motif
15
Q
Gradient of aurora
A
- Gradient of aurora A at spindle, B at centromere
- ↑ aurora A = kinetochore released from microtubule (at pole released + moves to middle
- Aurora B = near centromere, initial attachment = unstable due to aurora B
- Aurora B phosph NCD80 + KNL1, ↓ affinity for microtubule
- w/ tension, pull away, escape aurora B → dephosph
16
Q
Spindle checkpoint
A
- Check chromosome alignment
- Based on number of free kinetochores
- MAD/BUB regulate APC. Localise to kinetochores x attached to microtubules
17
Q
MPS1
A
- Senses + binds unattached kinetochores
- MPS1 phosph MELT in KLN1
- Aurora B phosph NDC80 → MPS1 binds phosph NDC80 → phosph MELT KLN1 → signals to SAC pathway
- Cdk activates MPS1 by phosph
18
Q
BUB3
A
- Reads phosph of KLN1
- Binds GLEBS motif in BUB1/BUBR1
19
Q
MAD2
A
- Open/closed conformation
- Close locks w/ Cdc20 + forms part of MCC
- MPS1 phosph. unattached kinetochore + recruits to checkpoint complex
- MPS1 phosph MAD1 which is in complex with Mad2c + recruits Mad2o
- Mad1-Mad2c-Mad2o-Bub1-Bub3-Cdc20 → MCC (Mad2c-BubR1-Bub3-Cdc20)
20
Q
APC/C regulation
A
- Cdc20 binds cyclin
- MCC inhibits APC/C by occupying substrate bs w/ BUBR1
- BUBR1 captured by 2nd CDC20 in MCC + MAD2
- At start of M, ACC phosph by cyclin B → active, binds Cdc20 when unattached kinetochore → inhibited by MCC
- MCC turnover by TRP13
- When kinetochores attached → MCC x made but is turned over so x inhibit ACC
21
Q
CDC2
A
- Required for cell cycle transitions
- Wee1 inhibits cyclin B → cyclin B accumulates in late G2 → activates Cdc25 → enters M
- Human G1/S transition is controlled by growth factors, yeast = nutrient
22
Q
Experiment
Identifying mammalian G1-S (cyclin E)
A
- Took human cDNA library + identified plasmid that complements Cln mutant
- Cyclin E identified
- Binds CDK-subunit + phosph histone
- Looking at northern blot mRNA of cyclin B, appears in regular manner
23
Q
CKI
A
- Identified w/ Y2H
- p16 binds CDK4/6 + prevents cyclin binding
- p27 blocks ATP binding
- Mitogens inhibit inhibitors p21/-27, release CDK-cyclin D
- p27/p21 cyclinD-CDK4 co-precipitate (needed for formation)
- Nuclear localisation
24
Q
p53
A
- Proteins of p27 family re-inhibit cyclin-CDK in response to stress
- Stress feeds to p53 TF
- Target of p53 = p21
- ↑ mutation in cancer
25
Q
Mitogen regulation of cyclin D- CDK
A
- Leads to ↑ responses in Ras/MAPK pathway
- Phosph TF like Ets → binds gene promoter of cyclin D
- cyclin D-p27-CDK4 (inhibit), is phosph by CAK, translocates to nucleus
- PI3K → p27 ↓ by SCF
26
Q
pRB
A
- pRB-E2F
- pRB-P (by cyclin D-CDK4) x bind E2F → binds gene promoters
- Cyclin E phosph Rb → amplification
- LoF
27
Q
Transcription cell cycle
A
- Cyclin E phosph cyclin D → destroyed by SCF
- Also phosph MYB, p-MYB → activates MuvB transcription of G2/M genes like cyclin A
- Cyclin A phosph cyclin E → destroyed by SCF
- Cyclin B destroyed cyclin A by APC
28
Q
Resetting the cell cycle APC
A
- Rb is dephosph on mitotic exit , re-bind E2F
- APC = active, destroys FOXM1
+ve/-ve feedback
29
Q
DNA replication only 1
A
- In M, origin of replication bound to DNA + phosph (inactive)
- Geminin sequesters CDT1
- In S, make PIC
- Geminin disappears at start of A
- Cdc6 disappears in late G1
- Narrow window in G1 where can trigger DNA synthesis
- CDT1 destroyed during DNA replication, Gemini uparrow in G2
30
Q
Cell cycle control. of centriole duplication
A
- Centrioles present in G1
- Centriole duplication regulated by PLK4
- PLK4 activity ↑, autophosph itself
- NEK2
31
Q
Environmental requirements for mammalian cell proliferation
A
- Yeast + bacteria = controlled by nutritional status
- Mammalian cells need nutrients, macromolecular nutrients + growth factors
32
Q
Quiescence
A
- W/o serum, cells withdraw → quiescence
- Comb of factors
- W/o serum arrest btw M + S in G0
- Re-enter into Go
33
Q
Restriction point
A
- GF act before restriction point to activate CDKs
- When pass x need GF anymore
34
Q
CDKs
A
- To progress through G1, cells require activation of G1 Cdks
- Later in G1, cyclinE/CDK2 are activated
- In S = cyclin A + B
35
Q
Growth factor receptors
A
- ↑ affinity
36
Q
Tyr kinase receptor
A
- Has single TM pass domain
- Insulin/insulin-like = exception
37
Q
Experiment
Is Tyr kinase required for mitogenic response
A
- Transfect w/ ‘novel’ RTK
- Treat cell w/ receptor
- Does activate ds signal
- See what part of receptor needs e.g. mutate kinase
38
Q
RTK activation
A
- Activation by dimerisation → brings together IC region of tyrosine kinase → phosph each other
- Associate w/ SH2
- Several effects (change localisation, phosphorylation, allosteric activation)
39
Q
Cooperativity of signalling pathways
A
- Mutate individual Tyr so x phosph
- No single pathway is responsible
40
Q
G-protein linked receptor
A
- E.g. endothelin
- 7TN, activates G protein
- GDP → GTP
- a subunits varied
- By activate P13K, PLC-y
41
Q
Cytokine receptor
A
- Assoc. w/ non-RTK like JAK
- Binding of receptor to cytokine stab. dimeric form
42
Q
Ser/Thr kinase receptor
A
- TGFB
- Ser/Thr kinase assoc w/ IC domain
- Heterodimer (TGFB)
43
Q
Notch receptor
A
- Long EC domain
- 2 proteolytic events
44
Q
PI3K signalling
A
- Heterodimer, p85 + p110
- p85 = SH2, SH3
- PIP2 → PIP3
- ds effector = Akt/Pkb
45
Q
PKB/Akt
A
- Ser/Thr kinase
- Use 2 structurally distinct inhibitors of PI3K + blocks PKB activity
- Thr in kinase domain T loop, Ser in CTD
- Mutation of either → Ala
- PDK1 phosph Thr
- PIP3 brings substrate to kinase, allows PDK1 to phosph Pkb
- mTORC2 phosph ser
- To activate PKB, need PI3K, PDK1, mTORC2
46
Q
Localisation
A
- PKB is activated at plasma membrane, disc + moves e.g. to nucleus
47
Q
PKB substrates
A
- Metabolism (GSK3 inhibited → GS activated), PFK2 stimulates glycolysis)
- Cell cycle control (GSK3 phosph cyclin D, inactivate GSK3 → cyclin D ↑, p21/p27)
- Cell growth (S6 kinase, CIF-4e, Aka inhibits TSC1/2, Rcb, TORC1, S6K phosph x inhibit 4E-BP)
- Cell survival/apoptisis (BAD-BclX, phosph to interact w/ 14-3-3, cell survives)
48
Q
Ras
A
- RTK phosph → assoc of GRB2 → SOS → Ras active → Raf → MEK
- Cross talk
- Myc ↑ expression of cyclin D, SCF subunit, ↑ E2F synthesis
49
Q
Coordination of growth + division
A
- Need to link
- Certain factor triggers cells to grow then divide once certain size (yeast) vs other factors cause cells to divide in mammals
50
Q
Yeast
Coordination of growth + division
A
- Divide at same size
- If overexposes cyclins, G1 phase is shortened, enter S phase faster → smaller
- WT cell in nutrient poor phase gives small cells,
51
Q
S pombe
Coordination of growth + division
A
- Wee1 inhibited by Cdt1, both in midsize
- Pom1 in poles
- Short cells Pom1 = pole + diffuses away, conc in midsize ↑
- Pom1 inhibits Cdt1 so x inhibit wee1 (Cdk phosph + inactive)
52
Q
S cerevisiae
Sense size
A
- Small bud forms on surface of mother
- Cell cycle inhibitor Whi5 (like Rb)
- ↑ size cell = ↓ conc of Whi5 until reaches threshold → S
- If overexpress Whi5 → bigger cells
53
Q
Uncoupled proliferation + growth
A
- Egg/neurons have withdrawn from cycle but still grow?
- Schwan cells
54
Q
Increased cell size + faster cell cycle entry
A
- ↑ size = faster entry
- Drosphilia overexposes Rheb ↑ size ↓ G1 so ↑ growth rate
55
Q
TOR
A
- Many nutrient sensing systems activate mTORC1
- Tor = either mTOCR1 or mTORC2
- mTORC1 inhibited by rapamycin, prevents substrate access
- mTORC1 = raptor, mTORC2 = rictor
- mTORC1 = activated ds of IGF1 receptor (IGFR → Act → TSC1/2 inhibits → inhibits Rheb → reg mTORC1)
- mTORC1 phosph S6K =, inhibits 4E-BP
- Akt inhibits Tsc1/2 → Rheb-GTP → mTOR active
- AMPK activates TSC1/2 → Rheb-GDP
56
Q
mTORC1 stimulation of cell growth
A
- S6K activation → phosph SKAR → activation of e1F4 factors → ↑ protein synthesis
- S6k → activates SREBP → ↑ glucose metabolism
- Autophagy inhibited
57
Q
mTORC2
A
- phosph Akt, ↑ substrates in cell cycle
- phosph PKC (cytoskeleton rearrangement)
- Activates SGK promotes ion transport
58
Q
Oncogenes
A
Genes that contribute in a dominant manner to transformation of a cell
- Can be virally encoded or modified cellular genes
59
Q
Proto-oncogene
A
- Normal cellular counterpart of an oncogene
- Mutation of protoco-oncogene → oncogene
60
Q
Tumour formation multistep
A
- Loss of tumour suppressor + activation of oncogene
- Loss of growth factor dependence
- Intensity to anti-growth signals
- Evasion of apoptosis
- Genomic instability
- Metabolic shift
- Immortality
- Sustained angiogenesis
- Metastasis
61
Q
Ongene activation
A
- Point mutation
- Amplification
- Chromosomal inversion
62
Q
Identification of oncogenes
A
- Tumour forming retroviruses
- Class 1 cause rapid formation of tumours after infection
- Multifunctional tumours
- Oncogenes encode constitutively active versions of proteins
- Virus picked up cellular genes
- e.g. capture of src by ALV
- Class II = slower onset
- Monoclonal tumours
- Virus integrates beside cellular gene
- myc 2 hotspots for insertion? - Isolation from tumour derived DNA
- Mutations in endogenous cellular genes
- Take DNA + introduce in growth-factor dependent cells, take GF away + see what grow
- A160 grow on top of one another - Chromosomal translocation
- E.g. Abl + Myc
- Can lead to removal of miRNA regulatory sequences e.g. translocations lead to loss of miRNA bs - Identification of genome amplification region
- e.g. erb2 brest cancer
- Use southern blotting to look at level
63
Q
Levels of oncogene action
A
- Growth factors
- E.g. sis - Growth factor receptor
- Leads to constitutive activation
- Activates ds signalling pathway, pass restriction point
- Mutation or overexpression (↑ chance of dimer) - Signalling pathway protein
- May only activate 1 branch
- e.g. V-src lacks inhibitory phosphate
- e.g. mutations that lock Ras in active state, residue 12 ↑ commonly mutated
e. g. PI3K, mutations along protein, more likely to happen - TF
- cycle progression needs new gene expression
- identify IE genes (add serum to quiescent cells, use RNAseq to see change in expression)
- Some IE genes are oncogenes which are themselves TF e.g. fos/jun
64
Q
Fos oncogene
A
- Rapidly induced
- Activation of v-fos involves deletion of ‘instability’ sequences in 3’ non coding
- Fos heterodimerises w/ Jun
- Viral Jun is truncated in regulatory region → stabilised
65
Q
Cyclin D1 expression
A
- Signalling through integrin → activation of Ras → MAPK active → Fos → Jun → Jun + Fos dimerise → ↑ expression of cyclin D1
- Also TFs like STAT, NF-Kb
66
Q
Myc
A
- Assoc w/ proliferation
- levels ↑ by GF
- Regulated by PTMs from other pathways e.g. phosphorylation by MAPK or GSK
- Switch btw proliferation + differentiation
- If myc lost, Max heterodimer w/ Mxt → repress mitogenic signals
- Directly activates transcription of Cdc25
- Inactivates p27 + p21
- ↑ cyclin D1 expression
67
Q
Mutations in Cdks
A
- Cyclin D = over expressed due to amplification
- mutations in cdk4 prevent INK4 binding
68
Q
Passenger and driver mutations
A
- Driver mutations → cancer
- Passenger mutations can be tolerated, often in introns
- Lung carcinoma has >50,000 singel nucleotide changes
69
Q
Oncogene cooperation
A
- Oncogenes + tumour suppressors work together
- ↑ genetic instability means pick up ↑ mutation
- E.g. overexpression of Myc and v-ras
70
Q
Cancer stem cell
A
- Controversial
- Certain cells in a tumour act like cancer stem cell
- Take cells in tumour, disc into single cells + re-introduce
- Small no. that can form tumour = stem cell like
71
Q
Drug treatment
A
- Previous = DNA damaging agents, ionising radiation
- Inhibit activated oncogenes
- Universal target like Ras, inhibit PTM
- Selective target e.g. Tamoxifen
- Kinase inhibitor
- ATP analogues x selective enough
- tyrosine kinase inhibitor = non-selective or specific inhibitors - Herceptin
- Monoclonal ab against Neu - Gleevec
- Inhibits Bcr-Abl
- Resistance due to point mutation - Targeting oncogene expression
- ↓ side effects
- Ribozyme can target BCR-Abl fusion function
- Delivery of ribozyme hard
Synergistic treatment
72
Q
Identification of tumour suppressor
A
- Fuse 1 tumour cell + 1 non tumour cell
- But hard to fuse, large parts of chromosome often lost - Look at familial traits
- Childhood cancer, inherit 1 WT + 1 mutant tumour suppressor from parent
- Aquire 2nd mutation (↑ chance), often gene conversion event - Clone genes that revert transformed cells
- Identify proteins that interact w/ immortalising oncogenes
- SV40 T antigen
73
Q
pRb
A
- Mutated, deleted or interaction w/ viral protein
- Associated w/ ↑ subset
- Re-introductino of WT pRb reverts transformed phenotype → returns to growth factor independence
- cyclinE hyperphosph pRB, pass restriction point
- viral oncogenes bind to pRB in pocket + block ability to interact w/ E2F
- Phosph sites can be mutated
- Histone deacetylases recruit E to inhibit transcription
- Comb of Rb + pRB-like proteins, their phosphorylation state + what E2F they associate w/
- Several targets (e2F → division), Suv39H1 senescence, let-2 differentiation
74
Q
p53
A
- Complete absence, mutated in both or just 1 allele
- 95% of mutations in DBD, x interact w/ DNA
- One of the most common genetic lesions
- TF, activated ds of stresses
- Can send cell to different response pathways, cell cycle arrest, apoptosis, senescence
- Li- Fraumeni
- Mdm2 inhibits (also oncogene, if ↑ activates p53), Arf sequesters Mdm2
75
Q
Activation of p53
A
- DNA damage
- Phosph by ATM protein kinase stab p53
- ssDNA damage activates ATR
- Chk1/2 → p53 phosph - Metabolic stress
- Glucose depletion activates AMPK, phosph p53
76
Q
ds of p53
A
- Cell cycle inhibition
- p21 (inhibits G1 CDK)
- 14-3-3 (sequesters Cdc25, x dephosphorylate CDK1) - Apoptosis
- ↑ levels of p53 → apoptosis
- Induces genes which ↑ ROS so ↑ Mit degradation + induces BH3 only PUMA - Angiogenesis
- Thrombospondin
77
Q
Targeting p53 in cancer
A
- Normally hard to target
- Dominant -ve nature means can use small molecule inhibitor
- E.g. Nutlins inhibits Mdm2
78
Q
pRb + p53 link
A
- p53-dependent G1/S arrest via p21 involves inhibitor of pRb phosph via Cdks
- Dereg E2F activity induces p19ARF + p53-dpeendent apoptosis
- P16INK4A + P19ARF = alternative reading frames of same locus (P16… = CDk4 inhibitor, p19.. stab p53
79
Q
Transcriptional activation of cell cycle
A
- +ve feedback (move to next stage and repress previous)
- pRb binds E2F to repress expression in G1, Cyclin D-Cdk4/6 phosph pRb
- E2F induces cyclin E expression → cyclin E further phosph pRb → transcribes cyclin A
- Cyclin A = -ve feedback, phosph E2F
80
Q
Degradation of mitotic proteins
A
- SCF
- Controls G1/S and G2/M boundaries
- SCF substrates = cyclin D/E + CKIs like p27 - APC
- Co-activators = Cdc20 + cdh1
- Activated cyclin B → activated Cdc20
- If free kinetochores, APC/C is inhibited by MCC
- MCC turned over by TRIP13 → free APC
- When kinetochores are attached to microtubules, ↓ formation of MCC but turnover of TRP13 carries on, ↓ inhibition on APC/C
- Cyclin B then degraded
81
Q
Regulation of proteolysis machinery
A
- APC/C needs Cdc20/Cdh1 to bind
- In early M, CDK phosph APC/C → Cdc20 binds → cyclins destroyed → ↓ CDK → anaphase
- Cdh1 is phosphorylated during S, G2 + M, x bind APC
- When exits M, Cdh1 is dephosph to activate APC. This ubiquit Cdc20 so x simultaneous activation of both
82
Q
CKIs
A
Yeast
- Sic1p phosph Cdc28-cyclin, prevents premature S phase entry
Mammals
- INK4 + CIP/KIP
- p27 highest in Go + early G1, ↓ ↓ in G1/S
- Prevent activation of cyclinE-Cdk2 or cyclinD-Cdk4
- E2F → p27 transcription → cyclin E/ cdk2 inhibited → x pRb phosph (-ve feedback)
83
Q
Regulation by phosphorylation
A
- Cdc2 = inhibited at Thr14/Tyr-15 + activated at Thr161
- Wee1/Cdc25
- CAK = activating phosph
- Cdk1 activates own activator Cdc25 + inhibits inhibitor, w/o cyclin B Cdk1 ↓, wee1 ↑ cdc25 ↓
84
Q
DNA replication control
A
- CDT1 needs to be free + dephosph
- Geminin sequesters CDT1 but destroyed by APC in A
- Cdt1 removed during DNA replication so PIC x re-assemble
85
Q
GFR as oncogene more
A
- Ligand binds to ectodomain → dimerisation → activates IC tyr kinase domain
- Phosphotyr activates ds components
- Point mutation to one that causes dimerisation + activated w/o ligand
- Or chromosomal translocation, replaces EC domain w/ segment that dimerises
86
Q
Intracellular transducer as oncogenes more
A
- Ras mutations in Q61, G12 + G13
- Q61 interfers w/ H20 coordination needed for nucleophilic attack
- G12/G13 prevent van der waal btw Ras + GAP through steric hindrance which affects orientation of catalytic Gly at 61
- Raf becomes oncogenic by mutation at Val600→ Glu600 (mimics activation loop)
87
Q
TF as oncogenes more
A
- Ras affects myc expression in 2 ways
1. P13K = ds of Ras, inhibits GSK3 → prevents Myc being proteolysed
2. Ras activates Raf-1 → activates MEK pathway → phosph of c-Myc - Myc = 40% of tumours
- ↑ effects e.g. cyclin D, CDKs (activates Cdc25), E2F (myc stimulates CDK → phosph pRb → frees E2F, directly w/ E2F promoter) + CKIs
88
Q
Oncogenes not involved in cell proliferation
A
- Bcl-2 inhibits apoptosis
- Tumour suppressors = also important e.g. p53
- Telomerase + VEGF also oncogenes
- Protein acts is important e.g. at branch vs in linear pathway
+ how easy to mutate
89
Q
p53 vs pRb
Activation
A
- p53 = phosph + stabilised by several kinases, Mdm2, ARF
- pRb = phosph
90
Q
p53 vs pRb
Effects - cell cycle
A
- pRb acts through E2F target genes, p53 acts directly via p53 target genes
- pRb binds E2F and blocks transcription (can repair damage)
- p53 acts through p21 (inhibit Cdk2, prevents inactivation by Prb)
- p21 null mice are deficient in cell cycle arrest
91
Q
p53 vs pRb
Effects - CIN
A
- Experiments knockout
- BubR1 can physically assoc. w/ + activate -53 during SAC checkpoint activation + p53 activates BUBR1
- pRb loss affects mitotic fidelity
92
Q
p53 vs pRb
Effects - Apoptosis
A
- Extrinsic
- Phosph pRb means genes like caspase 7 are expressed
- p53 induces expression of Fas TNF receptor - Intrinsic
- pRb induces MOMP by binding BAX
- p53 induces expression of pro-apoptic genes
93
Q
p53 vs pRb
Effects - metabolism
A
- E2F null mice
- p53 suppresses glucose transport directly by preventing GLUT1/4 transcription
- Oxidative metabolism
94
Q
p53 vs pRb
Effects - angiogenesis
A
- E2F target genes = bFGF
- Loss of pRb → activation of Id2
- p53 inhibits HIF
95
Q
Apoptosis initiation
A
Extrinsic pathway
- TM receptor-ligand or NK or cytotoxic T cell-mediated injection of granzymes
- Death receptor - ligand → IC domain recruits adaptor
- FADD recruits initiator caspases → form DISC → form executive caspases
Intrinsic pathway
- Bax/Bad inhibited by Bcl2/Xl but BH3 sequesters Bcl2.XL → free Bax/Bad to homodimerise → outer membrane pore
- Frees cytc which binds APAF1 + forms apoptosome → CARD domain recruits caspase 9 → activates executioner caspases like 3 + 7
- Smac/Diablo → AIF + endonuc G
96
Q
Apoptosis execution
A
Nuclear effects
- MOMP → AIF + endonuc G
- Caspase dependent = RAIDD + RIPK1 compete for binding to PIDD + lead to survival or apoptosis
- Caspase 2 = us of caspase 3 which cleaves ICAD → DNA fragment
- Nuclear lamina degraded by caspase
Cytoskeletal effects
- Caspases cleave ↑ components e.g. actin + myosin
- Caspase-mediated proteolysis of ROCK1
97
Q
Apoptosis degradation
A
- Phospholipid asymmetry + externalised PS
- Bridge molecules = phagocyte recognition ligand
- Some receptors bind directly to apoptotic cell
- Internalise particle
98
Q
Apoptosis regulation
A
- Bcl2/Xl block apoptosis by sequestering Bak/Bax
- Pro-survival genes must be overwhelmed
- Mit fission
- Bcl2 = regulated by p53
99
Q
Chromosome structure
A
- Loop scaffold model
- 20,000 fold compaction
100
Q
Nucleosomes
A
- Formed from 2 copies of 4 histone proteins
- 147 bp periodicity
101
Q
Histone tail modification
A
- H H4 lys-rich tail
- H H3 phosph on T3 by haspin kinase → Aurora B kinase recruited → phosphate H H3 on Ser10
- Δ charge by acetylation/phopsph → Δ compaction
102
Q
Chromosome scaffold
A
- Diffraction pattern of interphase nuclei show periodicity in reflections (2,11,30-40nm)
- Cryo-EM = chromatin disordered
103
Q
Do you need histones to make chromosomes
A
- Xenopus egg extract depleted of histone H1
- Still form chromosomes
104
Q
What is needed for chromosome condensation
A
- SMC1 = 1st SMC found by genetic screen
- Mutant x segregate DNA
105
Q
SMC function
A
- SMC1 + 3 = cohesin
- SMC1 + 3 = linked by regulatory subunit, rapidly binds + releases DNA
- Smc3 acetylation blocks wap1 release (S phase)
- Sororin inactivation by mitotic kinase → cohesin release
106
Q
Centromere
A
- Here, CENPA replaces H H3
- Also recruits that protect cohesion from kinases (Sgo1, PP2A)
- In P, arms of chromatid held as have cohesin
- Phosph by Cdk1/aurora B releases cohesin
- x happen at centromere as have PP2A
107
Q
Cohesin release
A
- Separase = protease for Scc1 of cohesin
- 2 regulatory partners (cyclin B + securin, inhibit)
- Once spindle checkpoint is satisfied, MPS1 forms MCC, inhibits APC
- When all kinetochores attached, TRP13 recycles Mad2, APC = active
- APC breaks down cyclin B + securing → separase x inhibited cohesin destroyed
108
Q
Condensin
A
- Made up of SMC2 + 4
- Sequentially condense chromosome
- In M, associates w/ DNA + extrudes loops of DNA through ring, makes DNA shorter
- Condensin 1 = 1st, CDK1
- Condensin II = 2nd, makes smaller loops
109
Q
High resolution analysis of chromosome structure
A
- Hi-C
- TADd lost at G2/M, replaced by diagonal band
- Remove condensin + see effects
110
Q
Cortical stiffness in anaphase
A
- Sea urchin eggs, how much suction needed
- Stiffness ↑ immediately after cell is cleaved
111
Q
Microtubules needed for cell cleavage
A
- Colchine inhibits M + cell cleavage if applied b4 A
- MT involved in deciding when cell cleaved
112
Q
Actin filaments needed for cell contraction
A
- Actin = disorganised in cells w/ cytochalasin
- Needed for cell division
- Acts w/ myosin
113
Q
Anaphase spindle formation
A
- Forms as cell exits Mit
- In metaphase, spindles capture chromosome
- In A, this ↑ in no. as ends are captured by PRC1 + MKLP1
- PCL1 = dimer, opposite orientation
- Assoc/ w/ KIF4A which transports PRC1 to + end of microtubule
114
Q
Control of anaphase spindle elongation
A
- PRC1 restricted to ‘tags’ at + end by KIF4A
- Narrow band of overlapping microtubule
115
Q
Aurora B localises to anaphase spindle
A
- Transported from chromatin to central spindle by kinesin motor MKPL2
- Phosph KIF4A + promotes PRC1 transport
116
Q
PLK1
A
- Localises to centrosome + anaphase spindle
- PRC1 = binding partner
- In A, PRC1 binds microtubules, becomes phosph by PLK (binds through PBD), PLK promotes cell cleavage
- Phosphatase removes CDK phosphorylation
117
Q
Protein phosphatases
A
- PP2A vs PP1
- PP2A = trimeric E
- B56 + B56 are inhibited in M by CDK
118
Q
MASTL
A
- Greatwall kinase in flies
- CDK1-cyclin B activates
- If deplete = delayed entry into M
- Greatwall is activated by CDK + in turn inhibits PP2A by phosph ENSA
- Cyclin B is destroyed → great wall inactivate → ENSA slowly dephosph → PP2A active → PRC1 dephosph
119
Q
PLK1 recruitment of ECT2 RhoGEF
A
- Centraspindlin = MLKP1 + CYK4
- Phosph by PLK1 + docks Rho GEF ECT2
- CDK phosph Ect2, centraspindlin + PCR1 is inactive
120
Q
How cells shape change controlled
A
- Rho regulate actin cytoskeleton
- Active RhoA recruit formin + RHOK → triggers actin polymerisation → RHOK targets mysoin
- Myosin generates force
121
Q
Mibody formation
A
- Formed during telophase
- Physical barrier
- Abscision = ESCRT-III
- CEP55 interacts w/ ESCRT III
- MLKP1 compressed to form nobody
