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MCB > cell cycle control > Flashcards

cell cycle control Flashcards

1
Q

what are the stages of cell division?

A

1- replication of chromosomes: S phase/DNA synthesis
2- segregation of chromosomes: M-phase, mitosis
3- division of cytoplasm: cytokinesis

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2
Q

how can you measure when DNA replication is occurring in a cell?

A

measuring rate of incorporation of radioactive nucleotides into the nucleus

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3
Q

how does the amount of DNA in the cell allow us to differentiate between phases of the cell cycle?

A

1- the amount of DNA is unchanged
2- doubling period where the amount of DNA in the nucleus doubles
3- amount of DNA in the nucleus remains the same
4- amount of DNA halves as chromosome segregation occurs before cytokinesis

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4
Q

what are the phases of the eukaryotic cell cycle?

A

G1, S, G2, M, cytokinesis

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5
Q

how long does G1 last?

A

10-12 hours

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6
Q

how long does S phase last?

A

6-8 hours

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7
Q

how long does G2 last?

A

3-4 hours

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8
Q

how long does mitosis last?

A

30 mins

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9
Q

what is G0?

A

a state of quiescence. not all the cells are dividing and have exited the cell cycle. occurs midway through G1 due to a checkpoint where environmental conditions determine cell division

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10
Q

how does the amount of DNA change at different points in the cell cycle?

A

G1 - 2n
S - 2n ->4n
G2- 4n
mitosis - 2n

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11
Q

what happens in prophase?

A

chromosomes condense, centrosomes move apart to the poles of the cells

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12
Q

what happens during pro metaphase?

A

nuclear envelope breaks down, cytoplasmic microtubules gain access to the chromosomes

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13
Q

what happens during metaphase?

A

chromosomes get pulled towards the equator by microtubules in the form of spindle fibres

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14
Q

what happens during anaphase?

A

sister chromatids are separated

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15
Q

what happens in telophase?

A

cell forms a contractile ring of actin-myosin in the middle of the cell to pinch the two cells off. the nuclear envelope reforms and chromosomes de-condense

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16
Q

why is it important that cell division is coupled with cell growth?

A

if they are not coupled, cells may get smaller and smaller at each cell division

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17
Q

why is drosophila a good model for studying cell cycle controls?

A

in the early embryos every stage is at the same stage of the cycle.
we have a good understanding of the genetics and mutations

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18
Q

why are cell divisions in culture problematic for study of cell cycle controls?

A

cells are asynchronous and an extract will not represent a single stage of the cell cycle

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19
Q

what drugs can be used to synchronise the cell cycle at S phase?

A

Aphidicoli, hydroxyurea, thymidine

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20
Q

what drugs can be used to synchronise the cell at M phase?

A

nocodazole

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21
Q

how does nocodazole work?

A

interferes with microtubule polymerisation, prevents mitosis from finishing

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22
Q

what is flow cytometry? what can it be used for?

A

a technique using a high pressure of jet liquid to force tiny droplets of fluid through a fluorescence detector. it is useful to detect when S phase has occurred by measuring the amount of DNA present

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23
Q

how did cell fusion experiments reveal the existence of dominant acting cell cycle regulators?

A
  • cells were arrested in G1 and G2 and mixed with a drug causing membrane fusion
  • when a mitotic cell was fused with a G1 cell, the G1 cell behaved as if it were mitotic - mitosis is dominant over other stages of the cell cycle
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24
Q

what conclusions were made based on cell fusion experiments?

A
  • mitotic cells contain an M-phase activator that will push the cell into mitosis
  • S phase cells contain S-phase activator that pushes G1 directly into S phase
  • G2 phase cells are insensitive to SPF,DNA does not re-replicate before mitosis (this is a critical control)
25
Q

what are SPF and MPF?

A

complexes of cyclin dependent kinases and cyclin

26
Q

what cdks are there in humans?

A

1,2,4,6 - a family of highly related serine/threonine kinases

27
Q

which cdk-cyclin complex control entry into mitosis (MPFs)?

A

cdk1-cyclinA/B

28
Q

which cdk-cyclin complex controls entry into s phase (SPF)?

A

cdk2/cyclin E

29
Q

what are the general features of cdks?

A
  • roughly 35kDa
  • kinase domains
  • N and C terminus, plus catalytic cleft
  • contains serine/threonine motifs
  • inactive conformation in the absence of a cyclin
30
Q

what are the general features of cyclins?

A

300-400AA in length

  • highly conserved cyclin box that binds to the cdk
  • N terminal destruction box controls destruction of these proteins
31
Q

how is activity of cdks controlled?

A
  • cdks are expressed constantly, but are only active when the relevant cyclin is present
  • each cyclin is subject to a cyclical pattern of synthesis and destruction during the cell cycle
32
Q

why are yeasts an ideal model organism for studying cell cycle control?

A
  • simple
  • eukaryotic but single celled
  • easy and cheap
  • usually haploid - ideal for loss of function mutations
  • cdks/cyclins conserved from yeast to humans
33
Q

how are conditional lethal mutants created?

A
  • random mutagenesis is performed using ionising radiation or mutagenic chemicals
  • if this causes loss of function to an essential gene, the yeast will die
34
Q

how were cell cycle controls identified in conditional lethal yeast mutants?

A
  • the same yeast are exposed to a mutagen and grown on two replica plates, one at permissive temperature and the other at restrictive temperature
  • colonies that grow at the permissive but not restrictive temperature contain conditional lethal mutants in essential genes
35
Q

how was wee1 identified as a cell cycle inhibitor?

A

loss of function experiments found wee1 mutants to be abnormally short - premature mitotic entry

36
Q

how were cdc2 and cdc25 identified as G2/M activators?

A

loss of function experiments found these mutants to be abnormally long - prevented mitotic entry

37
Q

how was the cdc2 gene cloned?

A

by complementation - it was found that at the restricted temperature, plasmids containing gene Y (cdc2) rescued the cell

38
Q

what did sequencing of cdc2, wee1 and cdc25 find out about these proteins?

A

cdc2 - encodes a protein kinase
wee1 - encodes a protein kinase
cdc25 - encodes a protein phosphatase

39
Q

what are the advantages of using frogs and marine invertebrates for cell cycle research ?

A
  • easily available in large quantities
  • exhibit synchronous cell divisions
  • large enough to be injected with proteins, extracts or DNAs
  • optically clear enough to visualise with light microscope
40
Q

what is meant by oocyte maturation?

A

once fertilised or given an electric shock or calcium, an egg develops in a mitotic state - this is reflective of a transition from G2 into mitosis

41
Q

what was significance of experiments involving introduction of cyclohgeximide into frog oocytes?

A

cycloheximide blocks protein synthesis. egg cells will not undergo division events
upon injection of MPF, they can go into the first division event - must be a component of MPF that requires synthesis

42
Q

how was MPF purified?

A

by fractionation of egg extracts to isolate active component
fractionation repeated until minimal sets of proteins were isolated
on a gel showed two bands 34kDa and 65kDa
34 kDa identified by antibodies against yeast cdc2 -CDK1
antibodies raised against sea urchin cyclin B identified 65Kda protein

43
Q

which cdk/cyclin complexes regulate G1?

A

cdk4/cyclin D

cdk6/cyclin D

44
Q

which cdk/cyclin complexes regulate S?

A

cdk2/cyclin E

cdk2/cyclin A

45
Q

which cdk/cyclin complexes regulate G2?

A

cdk1/cyclin A

46
Q

which cdk/cyclin complex regulates G2 to S?

A

cdk1/cyclin B

47
Q

why are cell cycle checkpoints needed?

A
  • to check for the correct conditions in the extracellular environment
  • check for DNA damage
  • check if replication is complete
  • check if the spindle is correctly formed
48
Q

what are the principles of cell cycle checkpoint control?

A

each checkpoint has a specific sensor, signalling and effector mechanism that allows it to prevent cell cycle progression

49
Q

what are the cell cycle checkpoints?

A

1- restriction checkpoint
2- DNA damage checkpoint
3- mitotic spindle checkpoint

50
Q

what does the restriction point checkpoint monitor?

A

extracellular conditions. e.g. presence of growth factors

51
Q

how does the restriction point checkpoint exert control?

A
  • exerts control through CKIs, particularly p16 family - these inhibit cdks 4&6
  • cyclin D expression is also stimulated by growth factor
  • high concentrations of growth factor block p16
52
Q

outline how the restriction point checkpoint responds to unfavourable cellular environment

A

sensor mechanism - senses decrease of stimulatory growth factors and receptor activation through EGFR. there is also an increase of inhibitory growth factors

signalling mechanism - down regulation of classical cell signalling pathways - decreased expression

effector mechanism - decreased abundance of cyclin D- inactivates cdk4/6. increased expression of p16
increased expression of p27

53
Q

what is the purpose of the DNA damage checkpoint?

A

DNA damage can be caused by internal and external factors. these can lead to mutations or breaks in the double helix. cells need to arrest the cell cycle to allow time for DNA repair

54
Q

how was the DNA damage checkpoint first identified?

A

in budding yeast: when exposed to DNA damage at high levels-
wild type: arrest and repair
repair mutants: arrest no repair, cells die
checkpoint mutants: don’t arrest in response to DNA damage

55
Q

how does the DNA damage checkpoint work?

A

sensor - protein recognises mismatches in base pairing
signalling - mutiple protein kinases transduce signal e.g. ATM, ATR, chk1, chk2. these are all ser/thr kinases
effector- stabilisation by phosphorylation of p53 leads to an increase in transcription of p21, expression of genes that promote apoptosis

56
Q

why is the mitotic spindle checkpoint at metaphase?

A

cdk1 must be activated to go into mitosis but inactivated to go out of mitosis. therefore, cyclins must be degraded to exit mitosis

57
Q

how does the mitotic spindle checkpoint work?

A

sensor: recognises if chromosomes are attached to poles of spindle by kinetochore microtubules
signalling: in the absence of full attachment to chromosomes, kinetochore sends signals to keep APC switched off
effector: prevents destruction of cyclins that would lead to mitotic exit

58
Q

what are causes of dysplasia in cancer cells?

A
  • loss of DNA damage checkpoints

- loss of mitotic spindle checkpoints

MCB (16 decks)

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