Regulation of the cell cycle Flashcards
Describe the basic cell cycle
underlain by interphase and the M phase
Describe interphase
- Gap 1 phase (sometimes containing the Gap 0 resting phase where the cell exits the cell cycle)
- S phase
- Gap 2 phase
What happens in the S phase of interphase?
- replication of the genome
- checks to ensure total and single replication occurs
Describe the M phase
- mitosis
- prophase
- prometaphase
- metaphase
- metaphase–anaphse transition
- anaphase
- telophase
- cytokinesis: cell division
Define mitosis
the equal separation of the genetic material into two daughter nuclei
Why must the cell cycle occur with high fidelity?
ensure the genetic identicality of daughter cells to parents
What does high fidelity mean in the mitotic context?
few errors in DNA copying and chromosome segregation
Describe perturbations to the cell cycle
mutation of proto-oncogenes resulting in inappropriate behaviour and triggering the development of cancerous cells and tumours
The rate maintenance of transcription and translation relative to size is controlled by
the nucleus
Describe cellular decision points
places in which there is an opportunity to halt or accelerate the progression
Give some cellular decision points
- G1-S boundary
- G2-M boundary
Describe the experimental approaches for regulator identification
- mammalian cell fusions
- embryonic cells (frogs, sea urchins)
- genetics (yeast, Drosophila)
- complementation and sequencing for orthology
Describe cell fusion experiments
uncover the dominant inducers of the cell cycle
How can dominance be established in cell fusion experiments
fusing two cells non-simultaneously derived that exist in different stages of the cell cycle
Describe the fusion of cells in S and G1 phases
- results in the activation of the S phase in the originally G1 nucleus
- S phase machinery is dominant to the G1 machinery
Describe the fusion of cells in the M phase and the G1 phase
- M phase is activated in the originally non-M nucleus
- dominance of M over non-M machinery
Describe decision points in egg maturation
mediated by MPF
MPF
- Maturation Promoting Factor / Mitosis Promoting Factor
- two subunits: a catalytic cyclin dependent kinase, and a regulatory cyclin B
- implicated in decision making and promoting maturation into mitosis
Describe the action of MPF in maturing eggs
- immature oocyte grows to complete Meiosis I
- ejects first polar body of meiosis products
- matures
- initiated by a progesterone stimulus, and carried out by MPF
- egg nucleus arrested in the initial metaphase of meiosis II (MPF highly active)
Describe mitosis post-fusion of the pronuclei
- zygote undergo several rapid mitotic cycles
- can be observed in Xenopus cells
Why do eggs arrest in metaphase of meiosis II?
relatively stable state of the condensed chromosomes
Describe what happens on experimental injection of mature egg cytoplasm from metaphase arrest into an immature oocyte in interphase arrest using a micropipette
results in Meiosis I completion, and the formation of a mature egg cell.
What ceases the metaphase arrest?
- fusion
- cyclin degradation begins
- facilitates congression of the cell cycle at the metaphase-anapahse transition
Describe cyclin degradation
- calcium induced
- cell cycle regulating
Describe cyclins
- e.g. cyclin A and cyclin B
- proteins that appear after fertilisation
- concentration changes during the cell cycle progression
- can be observed in sea urchins and surf clam proteomes under gel electrophoresis
Describe the initial function of cyclins
- activation of Cam-kinase II
- ejection of the second polar body
- completion of Meiosis II
Describe the initial mitotic divisions of the zygote
- omit G1 (including G0) and G2 phases
- in the essence of rapidity
Why must the egg be so large?
- omission of G1 and G2
- since division is not co-ordinated with growth, the sub-division cycles will each half the size of the egg
Describe the action of Mitosis Producing Factor
- not active in interphase
- spikes in activity increasing in concentration to metaphase
- decreasing to disappearance in anaphase
- functional conservation
Describe cdk
- cyclin-dependent kinase
- conserved across species
- isolated using cdc28-temperature-sensitive cells grown at 25 degrees Celsius
Describe the conservation of cdk
- between sea urchins and yeast, as cdc2
- not in frogs
Describe the general composition of cdk1 and cdk2
- N-lobe
- pre C-helix hairpin
- C-helix
- activation segment
- alpha-L12 domain
- Cks1 domain
- C-lobe
Describe the discovery that the dynamics of cdk and cyclin accumulation and degradation are integral to driving the cell cycle.
When transformed with cdc28 and grown at 35 degrees Celsius, the cells in colony were found to be non-synchronised in various stages of the cell cycle.
Describe cyclin and cdk in the cell cycle
- unbound in G2, mitotic cyclin can bind to Cdc2 producing M-phase promoting factor
- increase in cdk activity, and increase in cyclin B concentration, triggers mitotic onset
- dissociates after M phase
- proteolysis of cyclin B inactivates cdk, releasing Cdc2 back into the G1 phase, where it can bind the G1 cyclin to start kinase activity
- dissociation at the S phase boundary
Describe cdk2 in vivo
can perceived in its interactions with DHB on the nucleolar side using fluorescent tagging
DHB
DNA helicase B
Describe cdk2 activity across the cell cycle
- low in G1 (including the G0) phase; very little is extra-nuclear
- intermediate in the S phase (roughly equal spread between intra- and extra-nuclear DHB)
- at high cdk2 activity, very little DHB is intra-nuclear: most is phosphorylated in the cytoplasm
Describe cyclins and cdks across the cell cycle in mammalian systems
- in G1, G1 and S-cyclin have the highest activity, alongside cyclin D and cdk4 and -6
- in S, S-Cdk activity stays high, alongside cyclin A, E and cdk2 (the others drop)
- throughout G2 M-Cdk activity increases alongside cyclin B and cdk1; it stays high until the metaphase-anaphase boundary
- post-metaphase, there is little cyclin activity
Describe yeast model systems of mitosis
there is substrate specificity between a single cdk and multiple cyclins.
Describe cdk mechanics
- reversible phosphorylation of serine or threonine residues of its multiple target proteins
- uses a phosphate from ATP producing ATP, altering its properties allosterically, in order to co-ordinate the cell cycle
What are the functions of cdk
- regulation of chromatin structure
- spindle and kinetochore formation
- DNA replication
- cytokinesis
Describe the reverse cdk reaction
protein phosphatases release the molecule of inorganic phosphate on the residues.
Describe the mechanics of cdk activation
- partially activated by binding of the cyclin T-loop
- fully activated upon phosphorylation of the bound T-loop
Describe the effects of cdk activation
- decision point
- exponential increase in active kinase activating all substrates simultaneously, for an efficient transition
Describe cdk inactivation
- phosphorylation at an inhibitory site
- wee1 kinase
- reversed by cdc25 phosphatase
What happens if cdks become inhibited?
- cell cycle control is forgone
- cells enter mitosis prematurely
- become smaller and smaller
- can be perceived under fluorescence microscopy, where dominant ‘wee’ mutants cause hyperactivity of Cdc2 cells
Summarise phosphorylation control of cdk
- binding of M-cyclin to cdk1 creates inactivate M-Cdk
- action of CAK or wee1 creates an inactive M-Cdk with an inhibitory phosphate at the inhibitory region, and an activating phosphate and the activation site
- action of cdc25 results in active M-Cdk
- creates a positive feedback loop creating more inactive cdc25 phosphatase to be phosphorylated, and more wee1
CAK
cdk-activating kinase
wee1
cdk-inhibitory kinase
Describe CKI proteins
- block activity by wrapping around an active cyclin-Cdk complex
- used on dsDNA damage (such as through x-rays), enacted by the inactivation of S-cdk in the G1 or S phases
CKI proteins
- cdk inhibitor
- e.g. p27
Describe the cellular response to dsDNA damage
- ATM or ATR kinase activation
- Chk1 or Vhk2 kinase activation
- phosphorylating and activating p53, and releasing its Mdm2 group: stable and active and not degraded
- used to transcribe the p21 gene and translate this transcript, expressing p21
- wraps around an active G1-Cdks and S-Cdks, forming a complex and thus inactivating them
Describe p53
ubiquitylation and degradation of proteasomes
Describe p21
cdk inhibitor protein
