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Molecular Cell Biology > Cell Cycle (MY GOAT HIRO) > Flashcards

Cell Cycle (MY GOAT HIRO) Flashcards

1
Q

eukaryote replication forks

A

bidirectional replication forks emanate out from multiple origins on chromosome

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2
Q

Mitosis subphases

A

PMAT

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3
Q

Prophase

A

-Prophase:
chromosomes condense
spindles start forming
nuclear envelope degrades: prometaphase
allows interaction of spindles and chromosomes

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4
Q

metaphase

A

each chromosome connected to both poles
bipolar attachment

line up on metaphase plate

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5
Q

anaphase

A

separation of sister chromatids to either pole

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6
Q

telophase

A

chromosomes decondense
nuclear envelop starts forming
spindle begins depolymerising

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7
Q

cytokinesis

A

actin pinches off cell to make 2 separate daughters

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8
Q

atypical cell cycles:

A

No Gap phases:
early embryonic cleavage divisions

No cytokinesis:
Drosophila embryo syncytium

No mitosis:
Drosophila Polytene cells

no replication:
Meiosis
2 successive divisions without replication before division 2

cell cycle can be modified

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9
Q

Control of cell cycle in early embryo/fertilised egg

A

cleavage divisions with no mass increase
quick divisions
cells shrink each time

these divisions are more or less independent from their environment

so controlled mainly by internal signals

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10
Q

external signals for microorganisms

A

one main signal is nutrient availability

not enough = stop dividing
STATIONARY PHASE

depending on cell types
control system and mechanisms can differ

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11
Q

recognising M phase

A

easiest
PRESENCE OF CONDENSED CHROMOSOMES under the microscope
or absence of nuclear envelope

use DAPI stain to visualise DNA

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12
Q

Recognising S-phase

A

Under microscope all Interphase cells look alike
so use other methods

S-phase cells are replicating their DNA
so add labelled deoxynucleotides in the media
(H3-Thymidine, BrdU - detectable by Ab)

newly synthesised DNA in S-phase cells will incorporate label

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13
Q

recognising G1 vs G2

A

G2 cells have 2x as much DNA as G1 cells

can either stain with fluorescent DNA dye such as DAPI

and measure fluorescence on a camera

or use flow cytometry and get DNA content profile

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14
Q

DNA content profile from Flow cytometry

A

Suspend cells
DNA stain
some stain stronger depending on cell cycle phase

suspension drips through hole
machine shines light on drips
DNA dye fluorescence measured by camera
intensity reflects DNA content of cell

gives DNA content profile

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15
Q

interpreting DNA content profile

A

x axis: fluorescence i.e. relative DNA content

y axis: number of cells with this fluorescence value

see two peaks
one is twice as fluorescent as the other
G1 first
then G2 peak

S-phase cells in between - varying fluorescence levels depending on S-phase

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16
Q

Synchronous culture

A

normal cultures are Asynchronous
random mix of cell cycle stages at once

obtaining a Synchronous culture of cells at the same stage is crucial in research

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17
Q

Selection synchrony

A

Select particular stage of cells from asynchronous population
these cells will pass through cycle MORE OR LESS synchronously

done by either:
cell size:
-newly divided cells are small
select by centrifugation

mitotic wash-off:
-mitotic cells (mammalian culture) round up and loosely attach the surface
-can select them by shaking
-depends on cell type tho

DRAWBACK: Low yield

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18
Q

Induction synchrony

A

Start with asynchronous population
Impose cell cycle block
Release the block after some time

Benefit: High Yield of synchronised cells
cells also more closely synchronised

Drawback: can give potential artifacts due to manipulations.

eg induction synchrony to G1/S border:
-asynchronous
-Inhibit DNA synthesis by Hydroxyurea
-causes them to accumulate at G1/S border
-Remove HU after enough time
-culture now synchronous

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19
Q

Chemicals for induction synchrony before different stages

A

S - DNA synth inhibitors (HU, removing thymidine)

M - Spindle inhibitors (Colcemid, nocadazole)

G1 - Quiescence/Stationary:remove growth factors or nutrients

. - Conditional cell cycle mutants

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20
Q

Why are yeasts good genetic systems

A

-can grow as haploid - recessive phenotypes can be seen

-classical genetic analyses thorugh crosses

-range of molecular genetic manipulations possible

-entire genomes of some species sequenced + annotated

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21
Q

Budding yeast cell cycle

A

S. cerevisiae
4 phases
BUT
divide by budding
-G1: no bud
-S: small bud
-G2: mid size bud
-M: large bud

also: spindles begin to form in S-phase, unlike in human cells where they begin in M-phase

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22
Q

conditional mutants

A

eg Temperature sensitive
grow at permissive temp
cannot/die at restrictive (usually 37degrees)

can occur in any essential genes

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23
Q

Isolating temperature sensitive mutants

A

Mutagenise haploid yeast cells
incubate on plates at 23deg

blot this plate and make a replica on another
then incubate that at 37deg
temperature sensitive colonies disappear
-can map these back to colonies on original plate

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24
Q

Finding cell cycle mutants specifically

A

mutant will only need the mutated gene product at a certain cell cycle stage
so can progress through others fine but cannot pass through a certain one at restrictive temp

so put ts mutants at restrictive temp
cell cycle ts mutants will arrest at specific stage
can be recognised by morphology (eg no buds for G1)

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25
Q

How to analyse cell cycle mutants (cdc)

A

Phenotypic analysis

Classical genetics

molecular genetic analysis

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26
Q

Phenotypic analysis of cdc mutants

A

analysing the arrest stage of the mutants
look at:
-DNA contents
-Visualise state of nucleus, chromosomes, spindles
-biochemical analysis for proteins with cell-cycle functions

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27
Q

classical genetic analysis of cdc mutants

A

-making a diploid with a wt yeast to see if mutant is dominant or recessive
usually recessive

-complementation tests:
cross with other cdc mutants
if still temperature sensitive (no rescue) then both are mutants in the same gene

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28
Q

Molecular genetic analysis of cdc mutants

A

-Gene cloning:
physical isolation of the WT version of mutated gene
usually through complementation
-to isolate a DNA fragment which rescues the mutation

-Sequence
determine DNA sequence
>the predicted Amino acid sequence of gene product
>may give clue about biochemical function

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29
Q

what can we learn from cdc mutant analysis

A

proteins/gene products involved in particular cell cycle events

pathways which regulate particular cell cycle events

overall control of cell cycle progression

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30
Q

The START decision point

A

in the middle of G1
cell decides whether it will divide or do something else
-nutrition (if dividing w/out nutrition then cells would get smaller and smaller)
-cell size
-sexual signals (mating/meiosis)

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31
Q

Finding “Start”

A

withdraw some nutrients from an asynchronous culture:
-Cells in early G1 all arrest in G1
-cells in late G1,S,G2,M can all continue with the division cycle theyre currently in but then arrest in the next G1

Middle of G1 is the important boundary
START
-chekcpoint for nutrient availability
-if YES then keep going - Commited to division even if nutrients removed after
until they next reach the checkpoint

also check for size, sexual partner

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32
Q

Why the cell size requirement for start in S. cerevisiae

A

budding yeast
sometimes when a daughter cell buds off mother
the mother cell is large enough to continue right away in the cell cycle

BUT the daughter cell is too small so needs to wait until it reaches a larger size
so that subsequent cycles produce sufficiently large cells and they dont keep shrinking over time

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33
Q

Start as a developmental switch point (yeast)

A

cells meet partner - “courtship”
but the cell cycle continues until they reach the next start

at start - with the mating factor present - causes them to arrest at Start in G1

can then begin mating/conjugation

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34
Q

Cdc28 kinase

A

the key kinase for start (in S cerevisiae i think)

cdc28 mutants arrest at start in G1
normal cdc28 gene function required for passing start (ie commiting to division)

encodes a Serine/Threonine protein kinase
phosphorylates substrates leading to commiting to rest of cell cycle

is the equivalent of cdc2 in S. pombe

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35
Q

Checkpoint - emergency brakes

A

Hit yeast w X-ray
causes DNA damage
causes cell to arrest right before mitosis in S-phase

if a DNA break goes into mitosis
chromosomes separate and some chunk of chromosome gets lost

but if stop right before can allow for repair
then continue with intact DNA

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36
Q

Mutants defective in damage induced arrest process

A

Mutants who fail to arrest - radiation sensitive (rad mutants)

most rad mutants arrest in G2/M phase but then die as they cannot repair DNA

rad9 mutants can repair DNA fine
but lack the ability to arrest before M-phase so do not have time to repair before mitosis
and so die because of that

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37
Q

Multiple checkpoints within cell cycle

A

DNA damage: G2/M transition

DNA replication checkpoint: G2/M transition

Spindle assembly checkpoint: Metaphase/Anaphase transition, protects against trying to divide with eg spindle defects

and more

keep the cell cycle under control to ensure it occurs right each time

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38
Q

Benefits of Fission yeast (Schizosaccharomyces pombe)

A

-is yeast - easy to grow, manipulate, isolate mutants

-grow by length extension
cell length can be used as a marker for cell cycle

-are more similar to higher eukaryotes in some aspects
-mitosis more similar as spindle only forms in M-phase, and Cell division is symmetrical

39
Q

selecting cell cycle mutants for S. pombe

A

slightly different so S. cerevisiae

Cell cycle mutants become long
are able to grow at restrictive temp BUT it blocks division
so keep growing and become abnormally long

used to isolate a lot of cdc mutants

40
Q

more unusual S. pombe cell size mutants

A

wee mutants
go into mitosis early

in WT cell grows and enters mitosis at certain size

but wee mutants do this at smaller size - too early

41
Q

wee gene activity

A

are regulatory

cdc mutants can be any component missing
BUT only regulatory parts speed it up when broken

cdc mutants stop
wee mutants (regulatory) speed it up

42
Q

wee1 function

A

wee1
all mutants were recessive (loss of function)
meaning wee1 is a NEGATIVE regulator (an inhibitor) of mitosis entry

overexpression of wee1 delays or prevents entry into mitosis
gives the long cdc mutant phenotype w no mitosis

43
Q

wee2 function

A

wee2 mutant is a dominant allele of cdc2
a HYPERACTIVE version

hyperactivity of the gene causes early entry into mitosis so is a POSITIVE regulator (inducer) of mitosis

loss of function mutations of cdc2 gene delayed mitosis giving long cells

44
Q

third mitosis regulator: cdc25

A

recessive cdc25 mutants give arrest in G2 - long cells in S. pombe

overexpression gave the wee phenotype - early mitosis

so cdc25 is a positive regulator/inducer of mitosis

45
Q

how do wee1, cdc25 and cdc2 work together to regulate mitosis

A

wee1 (mitosis inhibitor) inhibits cdc2 activity

cdc25 activates cdc2 activity

cdc2 drives transition into mitosis

46
Q

Biochemical approach to mitotic entry - cell fusions - models

A

fused cell has 2 nuclei from 2 diff cells

fuse mitotic cell with interphase cell
either:
1. M cell has mitotic inducer present
fuse with I cell that does not
once fused the mitotic inducer can diffuse and induce mitosis in interphase nucleus

  1. mitotic inhibitor exists in interphase cell
    diffuses in fused cell
    mitotic chromosomes inhibited giving two interphase nuclei
  2. non-diffusible chromosome/nuclear factor:
    cant diffuse (maybe tightly associated w chromosomes)
    -no diffusion in fused cell
    mitotic and interphase chromosomes stay as they are

1 is correct - the DIFFUSIBLE MITOTIC INDUCER

47
Q

Biochemical approach 2 - using Xenopus oocytes

A

immature oocyte matures and begins meiosis
then arrests at metaphase of 2nd division

continues after fertilisation

-immature oocyte resembles G2 phase in cell
-mataphase of meiosis II resembles M phase in cell

can collect many of these cells as they are arrested at both of these points
(Meiosis only begins from immature oocyte when exposed to hormones)

M phase cytoplasm can induce maturation of the immature oocytes into “m-phase”

48
Q

The MPF (from the Xenopus oocyte experiment)

A

present in the cytoplasm of the meiosis II arrested oocyte

is able to auto-activate
so active MPF induces MPF-precursors in immature oocyte to become active MPF

49
Q

embryonic MPF assay (still xenopus stuff)

A

fertilise mature oocyte
can take cytoplasm from different stages of dividing embryonic cells
inject into immature oocyte and observe ability to induce maturation/MPF activation

MPF activation is high during mitosis
is when the inducing capabilities of the cytoplasm is highest
MPF activity is found late in G2 and in M-phase
is conserved in inducing mitosis AND meiosis

50
Q

MPF contexts

A

is a mitotic inducer

in different contexts:
Maturation promoting factor
Mitosis promoting factor
M-phase promoting factor

51
Q

Protein synthesis and mitosis in early embryonic cells

A

cleavage divisions during early ebryonic divisions
very little protein synthesis

BUT if it protein synthesis is inibited - no divisions take place
so this little bit of synth is important for cell division

52
Q

identifying the protein synthesised early embryo divisions important for cell division

A

introduce radioactive AAs to the early embryo cells to label any proteins currently being synthesised

run proteins from cell on gel

use autoragiograph to identify bands corresponding to the proteins synthesised during these divisions

discovered Cyclins
bands become stronger towards mitosis
peak
then come back down in interphase
-Cyclin A
-Cyclin B

53
Q

cyclin A and B synthesis and degradation pattern in xenopus embryonic cells

A

continuous synthesis during interphase
levels of the protein peak at beginning of M-phase
then degraded in M-phase - needed to exit mitosis

54
Q

mitotic inducers discovered in various systems

A

Fission yeast: cdc2 (and wee1/cdc25)

mammal: diffusible mitotic inducer

xenopus: MPF

sea urchins: cyclins

55
Q

the dual roles of S pombe cdc2 (cdc28 in s cerevisiae)

A

the same protein product controls Start and Mitosis
cdc2 (S. pombe) and cdc28 (s. cerevisiae) encode similar protein kinases
conserved
are functionally homologous
-expressing one species’ gene in a mutant of the other rescues the phenotype

56
Q

isolation of human cdc2 homologue

A

S. pombe cdc2 mutant
introduce a mixture of vectors with human cDNA
introduces the DNA into the yeast cdc2 mutants
then plate them

many recovered a random gene from the cDNA and were still temperature sensitive

however some were rescued by the human cdc2 homologue - were able to grow at restrictive temperature
-pick out colony
-sequence the cDNA that integrated

encodes a protein kinase similar to cdc2
called Hs cdc2 (homo sapiens)

ALL eukaryotes have a cdc2 homologue

57
Q

Purifying MPF from Xenopus oocytes

A

only knew that active MPF from unfertilised M-phase like egg could induce maturation in other cells

purify protein with same activity as MPF:
-homogenise oocytes
-fractionate the homogenate
-separetes protein according to size (big proteins fall faster)
-can inject different fractions into immature oocytes and look for maturation activity
-have narrowed down fraction containing MPF

-take this fraction and separate on another column
-can cycle this process
-gets purer and purer
-takes ages tho :/

58
Q

MPF heterodimer

A

consists of a heterodimer fo cdc2 and cyclin B xenopus homologues

this complex regulates G2/M transition in ALL eukaryotes

59
Q

cdk and cyclin families

A

cyclin dependent kinases:
requires cyclin subunit to be present to be catalytically active
cdc2 = cdk1

diff cyclins complex wit diff Cdks to form the binary kinase

60
Q

regulation of cdc2 activity

A

cdc2 levels are constant throughout the cell cycle
instead its kinase activity is regulated
by cyclin B levels going up and down

cdc2 kinase activity only happens when cyclin B levels go up
then decreases when cyclin B levels drop

though even when cyclin B is bound - Cdc2 activity still isnt very high
there is another level of control

61
Q

cdc2 activity regulation by wee1/cdc25

A

in all eukaryotes
cdc25 is an activator
wee1 is an inhibitor
of cdc2

cdc25 gene encodes a protein phosphatase
wee1 gene encodes a protein kinase
-phosphorylated cdc2 is inactive - wee1 product phosphorylates and inactivates it
-cdc25 phosphatase removes this Pi and allows it to activate

62
Q

wee1 and cdc25 activity in diff stages

A

wee1 activity higher in G2
so cdc2/cyclin B complex is inactive

cdc25 activity increases when M-phase begins
removes phosphate
allows cdc2/cyclin B to activate

cdc2/cyclin B activity inhibits wee1
so positive feedback once cdc25 activates it

cdc2/cyclin B complex can now phosphorylate substrates for beginning mitosis

63
Q

Yeast Cdk1 cell cycle regulation at diff stages

A

Cdk is the same at these stages but complexes w a different cyclin

Cdk1:G1-cyclin - progress past Start

Cdk1:G1/S-cyclin - takes part during G1

Cdk1:S-cyclin - progress into S-phase

Cdk1:Cyclin B(M-cyclin) regulates G2->M

64
Q

sequential action of different Cdk1 complexes during G1 (yeast)

A

Cdk1:G1-cyclin - phosphorylates targets which activate transcription of G1/S-cyclin and S-cyclin

G1/S-cyclin is active before S-cyclin
-because S-cyclin is usually inhibited
-BUT the G1/S-cyclin/Cdk1 complex destroys the S-cyclin inhibitor

65
Q

Cdk inhibitors

A

Cki
bind Cdk/cyclin complexes and inactivate them

the Cki inhibiting Cdk1/S-cyclin needs ti be destroyed before S-phase can commence
-Cdk1:G1/S-cyclin phosphorylates this Cki
-allows Cdk1/S-cyclin to be active and progress to S-phase

66
Q

protein degradation and cell cycle progress

A

in G1->S-phase progression:
-CKIs are degraded

in M-phase:
-Cyclin B is degraded to exit mitosis

67
Q

Ubiquitination

A

marks protein for degradation by the proteasome

ubiquitin chains added onto protein
once enough ubiquitin is attached, protein is now marked for degradation

Ubiquitin is added by ubiquitin ligase (E3)
selectively attach ubiquitin, determining specificity and timing of degradation

once chain is formed then localisation to proteasome for degradation is automoatic

68
Q

Anaphase promoting complex/Cyclosome (APC/C)

A

active only in M-phase and G1
ubiquitinaltes Cyclin A, B, other proteins

is an E3 which directs degradation during M-phase

69
Q

SCF

A

is an E3 to direct degradation during G1
active only in G1

ubiquitinates the phosphorylated CKIs from the Cdk1/S-cyclin complex
allowing it to be degraded

70
Q

Difficulties in cdk/ cell cycle studies in mammalian cells

A

yeast has just one Cdk
however mammals have multiple Cdks as well as mutliple cyclins
some cyclins also have multiple forms
>increases complexity of the system

also difficulty of genetic analysis:
-harder to KO in diploid mammal cell than in haploid yeast
-RNAi and CRISPR developments help w this
>but was harder in beginning

71
Q

determinor of Cdk/cyclin complex levels

A

limiting factor is cyclin levels
Cdk levels normally constant

so amount of cyclin determines the amount fo complex
and hence the activity of the corresponding Cdk

SO
overexpression of cyclins can be a way of determining their activity from the altering of the phenotype

72
Q

downside of overexpressing cyclins

A

can cause artifacts
can cause them to begin complexing with Cdks that it normally does not

73
Q

Overexpression of cyclins in mammals

A

Cyclin E: Shorter G1

Cyclin D: also shorter G1

Cyclins D and E are essential to G1->S progression

74
Q

Use of Ab to inactivate a cyclin

A

when there was no good method to KO a gene:

Inject Ab that binds and interferes with cyclin function (eg blocking Cdk binding)
-can use this to determine when different cyclins are important in cell cycle

Inject Cyclin D Ab: No DNA replication, cells stay in G1 - dont enter S-phase

evidence for cyclin D being essential for progress into S-phase

75
Q

Cdk function in yeast and parallels in Mammals

A

G1-Cdk complex: Cdk4, Cdk6 + cyclin D

G1/S-Cdk complex: Cdk2 + Cyclin E

S-Cdk complex: Cdk2 + Cyclin A

M-Cdk complex: Cdk1(=cdc2) + Cyclin B

76
Q

Quiescent cells

A

most adult cells not dividing
referred to as quiescent

metabolically active but not dividing
in terms of DNA content - are in G1 like state

some quiescent cells can be stimulated to divide
can switch on and off when needed

77
Q

cultured cell lines as model systems

A

cells taken from a person and put in culture:
-will all stop dividing after certain amount of divisions
-Senescence
-have to take cells many time

sometimes can get an immortalised cell line that can divide forever
-not 100% normal as have mutation that immortalises them
-but the cell cycle in some immortal cell lines are still normal so can be used for cell cycle research

78
Q

culture cells and growth factors

A

require them to divide
without them they enter a quiescent state

Serum usually added to culture media
contains multiple active components required for cell proliferation
-growth factors or mitogens

79
Q

Restriction Point

A

remove growth factors
if cell is in early G1
then they will stay in G1
dont go into S-phase or divide

if cell is in later G1 - cell still goes into S then divides (same for S, G2, M cells)
so within G1 there is a boundary
-before which cells need Growth factors to divide
-but after passing it they are commited to continuing through that cycle

this point is the Restriction point (R) in mammal cells
V similar to concept of Start in Yeast

80
Q

G0

A

say that cells in the quiescent state at R are in G0 not G1
as it can take hours to return to cell cycle after growth factors reintroduced

better to think of it as cell going into diff state outside of cell cycle

G0 molecularly different to G1

81
Q

2 separate controls of cell cycle

A

Cell cycle control:
-cell is going through cycle
-and then there is eg DNA damage
-cell stops there before it is fixed
>Mainly controlled through INTERNAL signals

Proliferation control:
-cell goes between being in cell cycle, or out of it (G0)
-mainly determined by external signals (growth factors…)

these two controls are linked at R point

82
Q

Mitogenic (growth factor) signal trasnduction pathway

A

-Growth factor binds Receptor Tyrosine Kinase
-activates Ras G-protein
-activates 3 kinases in sequence:
>Raf (MAP3K)
>MAP2K
>MAPK
-MAPK stimulation:
>stimulates general metabolism to grow cell
>AND transcription of cell cycle genes - triggers passage of R point

83
Q

Passing the R point

A

the MAPK
activates transcription of Cyclin D (unstable so only present with mitogen signalling), a G1 cyclin

complexes to form active Cdk4/6-Cyclin D

as soon as transcription stops:
-Cyclin D levels drop quick
-need high transcription to keep high Cyclin D levels

Cdk4/6-cyclin D complex actovates G1/S-Cdk and S-Cdk by activating trasncription of corresponding cyclins

84
Q

Activation of G1/S-Cdk and S-Cdk through Rb and E2F

A

Cdk4/6-Cyclin D (G1-Cdk) adds two phosphates to Rb protein
this inactivates it causing it to change conformation and free up the TF E2F

free active E2f can go to nucleus and activate transcription of Cyclin E and A
Allows for activation of G1/S-Cdk and S-Cdk

i think the same Cki stuff happens with active G1/S-Cdk and S-Cdk as in yeast

85
Q

in and out of cell cycle

A

in G0:
-Cdk and cyclin levels down
-Cki levels up
-assures cells are fully arrested

Transition from G0-G1:
-Cdks and cylcins resynthesised
-Ckis destroyed by SCF (a ubiquitin ligase/E3)
>this takes time - hence the lag for cells to exit G0 after serum added

86
Q

Transcriptional response from G0-G1

A

after serum re-added
2 waves of trancription
-Early response genes transcribed fast, within hours
-Delayed response genes transcribed later

Early response genes encode the TFs for delayed response genes
so if add a protein synthesis inhibitor:
-Early gene transcription is independent of protein synth
-however the transcribed mRNAs cannot be translated
-the TF products cannot go on to activate delayed response genes
-so no delayed response transcription

87
Q

Early response genes

A

MAPK phosporylates targets
those targets activate early response genes
early response genes encode TFs

88
Q

delayed response genes

A

actiavted by the TFs from early response
encode cyclin D
E2F
SCF subunits

components necessary for progression through G1 into S

89
Q

cancer + the cell cycle

A

clone of cells accumulating multiple mutations to gain anti-social behaviour
eg:
-uncontrolled proliferation
-invade other tissues
-survive and proliferate in foreign sites
-genetically unstable

cell cycel misregulation important part of cancer

90
Q

Cancer inducing genes

A

Proto-oncogenes
-present in normal cells
-can gain hyperactive mutations
-oncogene activated (induces cancer)
-these genes induce proliferation

Tumour suppressor genes:
-loss of function mutation happens in gene that normally inhibits proliferation
-induces cancer
-usually recessive mutation need both loss of function alleles

many anti cancer drugs target cell cycle proteins

91
Q

Proto oncogene examples

A

Growth factors
RTKs
Ras G-protein
early response gene TFs
Cyclin D

92
Q

tumour suppressor gene examples

A

Rb
CKIs

93
Q
A
94
Q
A

Molecular Cell Biology (5 decks)

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