Cell Cycle (MY GOAT HIRO) Flashcards
eukaryote replication forks
bidirectional replication forks emanate out from multiple origins on chromosome
Mitosis subphases
PMAT
Prophase
-Prophase:
chromosomes condense
spindles start forming
nuclear envelope degrades: prometaphase
allows interaction of spindles and chromosomes
metaphase
each chromosome connected to both poles
bipolar attachment
line up on metaphase plate
anaphase
separation of sister chromatids to either pole
telophase
chromosomes decondense
nuclear envelop starts forming
spindle begins depolymerising
cytokinesis
actin pinches off cell to make 2 separate daughters
atypical cell cycles:
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
Control of cell cycle in early embryo/fertilised egg
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
external signals for microorganisms
one main signal is nutrient availability
not enough = stop dividing
STATIONARY PHASE
depending on cell types
control system and mechanisms can differ
recognising M phase
easiest
PRESENCE OF CONDENSED CHROMOSOMES under the microscope
or absence of nuclear envelope
use DAPI stain to visualise DNA
Recognising S-phase
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
recognising G1 vs G2
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
DNA content profile from Flow cytometry
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
interpreting DNA content profile
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
Synchronous culture
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
Selection synchrony
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
Induction synchrony
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
Chemicals for induction synchrony before different stages
S - DNA synth inhibitors (HU, removing thymidine)
M - Spindle inhibitors (Colcemid, nocadazole)
G1 - Quiescence/Stationary:remove growth factors or nutrients
. - Conditional cell cycle mutants
Why are yeasts good genetic systems
-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
Budding yeast cell cycle
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
conditional mutants
eg Temperature sensitive
grow at permissive temp
cannot/die at restrictive (usually 37degrees)
can occur in any essential genes
Isolating temperature sensitive mutants
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
Finding cell cycle mutants specifically
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)
How to analyse cell cycle mutants (cdc)
Phenotypic analysis
Classical genetics
molecular genetic analysis
Phenotypic analysis of cdc mutants
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
classical genetic analysis of cdc mutants
-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
Molecular genetic analysis of cdc mutants
-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
what can we learn from cdc mutant analysis
proteins/gene products involved in particular cell cycle events
pathways which regulate particular cell cycle events
overall control of cell cycle progression
The START decision point
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)
Finding “Start”
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
Why the cell size requirement for start in S. cerevisiae
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
Start as a developmental switch point (yeast)
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
Cdc28 kinase
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
Checkpoint - emergency brakes
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
Mutants defective in damage induced arrest process
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
Multiple checkpoints within cell cycle
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
Benefits of Fission yeast (Schizosaccharomyces pombe)
-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
selecting cell cycle mutants for S. pombe
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
more unusual S. pombe cell size mutants
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
wee gene activity
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
wee1 function
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
wee2 function
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
third mitosis regulator: cdc25
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
how do wee1, cdc25 and cdc2 work together to regulate mitosis
wee1 (mitosis inhibitor) inhibits cdc2 activity
cdc25 activates cdc2 activity
cdc2 drives transition into mitosis
Biochemical approach to mitotic entry - cell fusions - models
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
- mitotic inhibitor exists in interphase cell
diffuses in fused cell
mitotic chromosomes inhibited giving two interphase nuclei - 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
Biochemical approach 2 - using Xenopus oocytes
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”
The MPF (from the Xenopus oocyte experiment)
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
embryonic MPF assay (still xenopus stuff)
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
MPF contexts
is a mitotic inducer
in different contexts:
Maturation promoting factor
Mitosis promoting factor
M-phase promoting factor
Protein synthesis and mitosis in early embryonic cells
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
identifying the protein synthesised early embryo divisions important for cell division
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
cyclin A and B synthesis and degradation pattern in xenopus embryonic cells
continuous synthesis during interphase
levels of the protein peak at beginning of M-phase
then degraded in M-phase - needed to exit mitosis
mitotic inducers discovered in various systems
Fission yeast: cdc2 (and wee1/cdc25)
mammal: diffusible mitotic inducer
xenopus: MPF
sea urchins: cyclins
the dual roles of S pombe cdc2 (cdc28 in s cerevisiae)
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
isolation of human cdc2 homologue
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
Purifying MPF from Xenopus oocytes
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 :/
MPF heterodimer
consists of a heterodimer fo cdc2 and cyclin B xenopus homologues
this complex regulates G2/M transition in ALL eukaryotes
cdk and cyclin families
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
regulation of cdc2 activity
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
cdc2 activity regulation by wee1/cdc25
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
wee1 and cdc25 activity in diff stages
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
Yeast Cdk1 cell cycle regulation at diff stages
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
sequential action of different Cdk1 complexes during G1 (yeast)
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
Cdk inhibitors
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
protein degradation and cell cycle progress
in G1->S-phase progression:
-CKIs are degraded
in M-phase:
-Cyclin B is degraded to exit mitosis
Ubiquitination
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
Anaphase promoting complex/Cyclosome (APC/C)
active only in M-phase and G1
ubiquitinaltes Cyclin A, B, other proteins
is an E3 which directs degradation during M-phase
SCF
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
Difficulties in cdk/ cell cycle studies in mammalian cells
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
determinor of Cdk/cyclin complex levels
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
downside of overexpressing cyclins
can cause artifacts
can cause them to begin complexing with Cdks that it normally does not
Overexpression of cyclins in mammals
Cyclin E: Shorter G1
Cyclin D: also shorter G1
Cyclins D and E are essential to G1->S progression
Use of Ab to inactivate a cyclin
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
Cdk function in yeast and parallels in Mammals
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
Quiescent cells
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
cultured cell lines as model systems
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
culture cells and growth factors
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
Restriction Point
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
G0
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
2 separate controls of cell cycle
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
Mitogenic (growth factor) signal trasnduction pathway
-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
Passing the R point
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
Activation of G1/S-Cdk and S-Cdk through Rb and E2F
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
in and out of cell cycle
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
Transcriptional response from G0-G1
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
Early response genes
MAPK phosporylates targets
those targets activate early response genes
early response genes encode TFs
delayed response genes
actiavted by the TFs from early response
encode cyclin D
E2F
SCF subunits
components necessary for progression through G1 into S
cancer + the cell cycle
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
Cancer inducing genes
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
Proto oncogene examples
Growth factors
RTKs
Ras G-protein
early response gene TFs
Cyclin D
tumour suppressor gene examples
Rb
CKIs
