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Cell growth + division ALL Flashcards

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1
Q

Stages of cell cycle

A
  • G1, S, G2, M
  • Microscopy = useful for describing events
  • Yeast = genetic / mechanistic info
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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
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3
Q

Experiment

Control of cell cycle

A
  • Temp sensitive mutant of CDC (grow at ↓ not ↑ temp)

- Find what complements cdc mutant

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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)
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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
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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
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7
Q

Cyclin degradation

A
  • Mutagenesis showed 1st 90 aa of cyclinB has D-box, recognised by ubiquitin
  • Ubiquitin recognised by proteasome
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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
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9
Q

Tubulin

A
  • Building block of spindle
    • and 0 end
  • If meets chromosome, becomes stabilised
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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
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11
Q

Kinesin + dynein

A
  • Force generating ATPases

- Move chromosomes

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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
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13
Q

Spindle attachment geometry

A
  • Correct = amphitelic
  • Incorrect = monotonic, systelic + merotelic
  • Need way of sensing
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14
Q

Aurora kinases

A
  • Adopt similar active conformation to cyclin bound to Cdk?

- Phosph to stabilise AS and have nearly identical consensus motif

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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
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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
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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
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18
Q

BUB3

A
  • Reads phosph of KLN1

- Binds GLEBS motif in BUB1/BUBR1

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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)
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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
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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
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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
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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
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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
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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
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26
Q

pRB

A
  • pRB-E2F
  • pRB-P (by cyclin D-CDK4) x bind E2F → binds gene promoters
  • Cyclin E phosph Rb → amplification
  • LoF
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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
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28
Q

Resetting the cell cycle APC

A
  • Rb is dephosph on mitotic exit , re-bind E2F
  • APC = active, destroys FOXM1
    +ve/-ve feedback
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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
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30
Q

Cell cycle control. of centriole duplication

A
  • Centrioles present in G1
  • Centriole duplication regulated by PLK4
  • PLK4 activity ↑, autophosph itself
  • NEK2
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31
Q

Environmental requirements for mammalian cell proliferation

A
  • Yeast + bacteria = controlled by nutritional status

- Mammalian cells need nutrients, macromolecular nutrients + growth factors

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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
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33
Q

Restriction point

A
  • GF act before restriction point to activate CDKs

- When pass x need GF anymore

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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
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35
Q

Growth factor receptors

A
  • ↑ affinity
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36
Q

Tyr kinase receptor

A
  • Has single TM pass domain

- Insulin/insulin-like = exception

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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
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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)
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39
Q

Cooperativity of signalling pathways

A
  • Mutate individual Tyr so x phosph

- No single pathway is responsible

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40
Q

G-protein linked receptor

A
  • E.g. endothelin
  • 7TN, activates G protein
  • GDP → GTP
  • a subunits varied
  • By activate P13K, PLC-y
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41
Q

Cytokine receptor

A
  • Assoc. w/ non-RTK like JAK

- Binding of receptor to cytokine stab. dimeric form

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42
Q

Ser/Thr kinase receptor

A
  • TGFB
  • Ser/Thr kinase assoc w/ IC domain
  • Heterodimer (TGFB)
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43
Q

Notch receptor

A
  • Long EC domain

- 2 proteolytic events

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44
Q

PI3K signalling

A
  • Heterodimer, p85 + p110
  • p85 = SH2, SH3
  • PIP2 → PIP3
  • ds effector = Akt/Pkb
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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
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46
Q

Localisation

A
  • PKB is activated at plasma membrane, disc + moves e.g. to nucleus
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47
Q

PKB substrates

A
  1. Metabolism (GSK3 inhibited → GS activated), PFK2 stimulates glycolysis)
  2. Cell cycle control (GSK3 phosph cyclin D, inactivate GSK3 → cyclin D ↑, p21/p27)
  3. Cell growth (S6 kinase, CIF-4e, Aka inhibits TSC1/2, Rcb, TORC1, S6K phosph x inhibit 4E-BP)
  4. Cell survival/apoptisis (BAD-BclX, phosph to interact w/ 14-3-3, cell survives)
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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
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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
  1. Loss of growth factor dependence
  2. Intensity to anti-growth signals
  3. Evasion of apoptosis
  4. Genomic instability
  5. Metabolic shift
  6. Immortality
  7. Sustained angiogenesis
  8. Metastasis
61
Q

Ongene activation

A
  • Point mutation
  • Amplification
  • Chromosomal inversion
62
Q

Identification of oncogenes

A
  1. 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?
  2. 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
  3. Chromosomal translocation
    - E.g. Abl + Myc
    - Can lead to removal of miRNA regulatory sequences e.g. translocations lead to loss of miRNA bs
  4. Identification of genome amplification region
    - e.g. erb2 brest cancer
    - Use southern blotting to look at level
63
Q

Levels of oncogene action

A
  1. Growth factors
    - E.g. sis
  2. Growth factor receptor
    - Leads to constitutive activation
    - Activates ds signalling pathway, pass restriction point
    - Mutation or overexpression (↑ chance of dimer)
  3. 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
  4. 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
  1. Kinase inhibitor
    - ATP analogues x selective enough
    - tyrosine kinase inhibitor = non-selective or specific inhibitors
  2. Herceptin
    - Monoclonal ab against Neu
  3. Gleevec
    - Inhibits Bcr-Abl
    - Resistance due to point mutation
  4. 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
  1. Fuse 1 tumour cell + 1 non tumour cell
    - But hard to fuse, large parts of chromosome often lost
  2. Look at familial traits
    - Childhood cancer, inherit 1 WT + 1 mutant tumour suppressor from parent
    - Aquire 2nd mutation (↑ chance), often gene conversion event
  3. Clone genes that revert transformed cells
  4. 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
  1. DNA damage
    - Phosph by ATM protein kinase stab p53
    - ssDNA damage activates ATR
    - Chk1/2 → p53 phosph
  2. Metabolic stress
    - Glucose depletion activates AMPK, phosph p53
76
Q

ds of p53

A
  1. Cell cycle inhibition
    - p21 (inhibits G1 CDK)
    - 14-3-3 (sequesters Cdc25, x dephosphorylate CDK1)
  2. Apoptosis
    - ↑ levels of p53 → apoptosis
    - Induces genes which ↑ ROS so ↑ Mit degradation + induces BH3 only PUMA
  3. 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
  1. p53-dependent G1/S arrest via p21 involves inhibitor of pRb phosph via Cdks
  2. Dereg E2F activity induces p19ARF + p53-dpeendent apoptosis
  3. 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
  1. SCF
    - Controls G1/S and G2/M boundaries
    - SCF substrates = cyclin D/E + CKIs like p27
  2. 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
  1. Extrinsic
    - Phosph pRb means genes like caspase 7 are expressed
    - p53 induces expression of Fas TNF receptor
  2. 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

Paper 4 (31 decks)

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