Cell cycle

What are the 3 events ensured by the cell cycle?

• chromosome replication

• chromosome segregation

• cell division

What triggers normal cells to proliferate? What could halt this?

extrinsic factors

Other signalling proteins can overrule these stimulatory factors and force a halt to proliferation = post-mitotic, differentiated state

Where does the cell cycle clock operate? What are the 3 outcomes of signals integrated by the cell?

in the nucleus

proliferation, quiescence, differentiation

What are the 4 main phases of the cell cycle and the outcome?

G1, S, G2, mitosis, cytokinesis

What are the 5 stages of mitosis and the outcome?

prophase, prometaphase, metaphase, anaphase, telophase

cytokinesis

What happens during G1 phase of interphase?

• cell increases in size

• ribosomes, RNA produced

• preparation for DNA synthesis

What happens during S phase of interphase?

DNA synthesised

What happens during G2 phase of interphase?

cell checks fidelity of DNA and preparation fro nuclear division

What happens during mitosis?

cell division

How long does a cell take to be regenerated ie for cell cycle to occur in entirety? How long does each phase last?

22 hours

G1: 10, S: 7.5, G2: 3.5, M: 1

What are different types of checks that can happen during the cell cycle?

DNA damage, stalled replication forks, unduplicated DNA, chromosome attachment to mitotic spindle, favourable environmental conditions

At what points of the cell cycle do the main checkpoints occur?

• G1 if favourable env. conditions then S phase, if not back into G0 or apoptosis

• G2 checks for DNA damage before entering M

• M, spindle checkpoint during metaphase, check chromosomes are attached to spindle fibres

What is the R point in cell cycle? When does it occur?

point of no return called the restriction point that happens at the end of G1, preparing the cell for S phase, serum and growth factors removed in the final hr of G₁ still lead to mitosis

What are the 2 decisions that can be made by G1 before R point? What is this dependent on?

• revert to G0 - Serum and growth factors removed before the cells have completed 80-90% of G₁

• proceed to S, G2, M phase

env. conditions

Considering late G1 to M is similar in normal and cancer cells, how is R point related to cancer?

Deregulation of the R-point decision-making machinery accompanies the formation of most types of cancer cells

What are a genetic or biochemical approach to finding a useful model system?

• genetic: cells that have a mutation in a putative cell cycle transition gene

• biochemical: Requires supply of large numbers of cells undertaking the same transition at the same time

What are the 2 types of yeast division?

• fission yeast - divides into two daughter cells through the centre

• budding yeast - daughter cell buds off the mother cell leading to 2 cells

What are the advantages of yeast as a genetic model for cell cycle?

Rapid division rate <1hr

Cell cycle control genes are HIGHLY conserved

Yeast can be grown as haploids or diploids

Easy to grow

You can tell which phase of the cell cycle the yeast cell is in by just looking (the length of the cell)

What does a Cdc gene stand for?

cell-division-cycle gene

What do temperature sensitive mutations allow in terms of cell growth?

growth at permissive temperature (low) but NOT HIGH

What is "cell-free mitosis"? What can it allow?

combining in vitro cytoplasm from frog eggs with nuclei from frog sperm eg = mitosis in a tube

• deplete the cytoplasm of different proteins using antibodies

• remove cytoplasm at different stages to study changes 

  (eg in protein phosphorylation) over time. 

What’s the weight of a CDK?

32 DD

What are CDKs regulated by?

Tyr phosphorylation and cyclin binding

How was cdc2/CDK1 ID’d? What does it encode?

yeast genetics

p34cdc2: changing the state of p34cdc2 it is possible to reprogramme a cell from entering mitosis to undergoing S phase

How do protein kinases operate to create molecular switches?

signalling devices that can phosphorylate a target protein, turning it on // turned off by phosphatase

Tyrosine, Serine and Threonine can be phosphorylated to modify their function, how is this written?

P-Tyr, P-Ser, P-Thr = regulation of cellular activity

Does mitosis correlate with kinase “on“ or “off“?

“on“

How can we visualise and quantify kinase activity?

• selectively extract kinase

• incubate it with a protein substrate and ATP

• electrophoresis of substrate and imaging

Do cell cycle transitions involve irreversible or reversible destruction of cyclins? At which stage are kinases inactive? Cyclin bound to kinase? And cyclins?

irreversible

G1, S, G2

M

destroyed from M to G0

In yeast, how many types of cell cycle regulator kinase genes are there? What are they called? And cyclins? How does this compare to mammals?

1 called CDK1 for multiple cyclins

Multiple CDKs and cyclins

When is cyclin E lowest? Highest?

through G1

after R point

When is cyclin A lowest? Highest?

all cycle except S phase

S phase

When is cyclin B lowest? Highest?

G1 and S

G2 and M

How do cyclin levels keep dropping through the cell cycle? What does this mean about the progression of the cell cycle?

ubiquitination-dependent degradation

can only progress in one direction

What are the 2 major control factors of cell cycle?

(1)Cyclins/CDKs– this drives the cycle forward 

(2) Mechanisms to stop the cycle (and correct) if there are problems ie Checkpoints

Which type of cyclins are controlled by extracellular signals? How many types are there in mammals?

D type

D1, D2, D3

What are Cyclin/CDKs regulated by?

CDK inhibitors (CKIs)

What’s the cdk partner for cyclin A, B, E in vertebrates?

B: cdk1

A: cdk1 and cdk2

E: cdk2

What’s the cdk partner for cyclin D in vertebrates?

Cdk4 and 6

What are the cdk partners for cyclins cln1,2 ; clb5, 6 ; clb1,2,3,4, cln3 in budding yeast??

all cdk1 bc only one type in yeast

What strategy was used to purify and identify cell cycle regulators? Which factor was identified as a regulator? By who?

studying early embryonic cells = Something in the Egg cytoplasm can catalyse the transition from G2 to M-phase 

Maturation Promoting Factor (MPF) by Yoshio Masui

When are arrest signals sent in embryo meiosis stages? What are the stimuli?

• just before meiosis 1 in G2 then in metaphase 2 of M phase

• Maturation stimulus MPF - releases interphase arrest // sperm entry releases mitotic arrest