lecture 4 - Cell Cycle Control

Question 1

Which cells divide in animals, e.g., humans?

Answer 1

Question 2

Control of the cell cycle

Answer 2

contact inhibition

Question 3

Control of the cell cycle – physical mechanisms

Answer 3

Timing of cell division in different parts of the body are carefully controlled by Chemical & Physical mechanisms.

Anchorage dependence: they must be in contact with a solid surface.

Density-dependent inhibition: Crowded cells stop dividing. Animal cells stop dividing when they touch each other.

Question 4

What are the main drivers? Cell fusion experiments

Answer 4

Question 5

The African clawed frog (Xenopus laevis) – oocyte maturation

Answer 5

Question 6

Discovery of maturation promoting factor MPF

Answer 6

Cytoplasm taken from mature oocytes & injected into G2 immature oocytes induces oocytes to enter the first meiotic division. M phase

Cytoplasm taken from a variety of actively dividing cells from a range of organisms could prematurely induce M phase

MPF activity low in G2 & peaked in mitosis.

MPF was purified - a protein with two subunits, and had kinase activity

Question 7

Discovery of cyclins: Sea urchin

Answer 7

Sea urchin eggs can be synchronised in their early cell division.

Proteins discovered that rise and fall with the cell cycle - called cyclins

Question 8

Genetic analysis of the cell cycle using brewer’s yeast

Answer 8

Question 9

How are conditional mutants used to identify genes involved in the cell cycle?

Answer 9

• A mutagen is used to create conditional mutants—cells that grow normally at one temperature (e.g. 22–23 °C) but are blocked in cell division at a restrictive temperature (e.g. 35 °C).
• These mutants aren’t dead but become elongated due to failure to divide.
• This approach led to discovery of several cdc (cell division cycle) genes, such as cdc2, which is the yeast equivalent of cdk1 in humans, and has kinase activity critical for cell cycle progression.

Question 10

Cyclins and Cyclin Dependent Kinases

Answer 10

Cyclin dependant kinase (cdk) a protein kinase enzyme only active when complexed with cyclin

Cyclin: the regulatory subunit of the dimer. Synthesised and destroyed in the cell cycle.

In MPF, which stimulates mitosis, the cyclin is called Cyclin B, or M cyclin

Question 11

The three main classes of cyclins

Answer 11

G1/S-cyclins: trigger progression through start, resulting in commitment to cell cycle entry.
S-cyclins: bind to Cdks soon after progression through start and help stimulate chromosome duplication.
M-cyclins: activate Cdks to stimulate entry into mitosis.
Yeast only have one Cdk, mammals have several.

Question 12

Why does M-Cdk activity increase suddenly, even though Cyclin B levels rise gradually?

Answer 12

• Although Cyclin B accumulates gradually, M-Cdk (Mitosis-promoting Cdk) activity rises sharply due to additional layers of regulation.
• Studies using two mutant strains of S. pombe (fission yeast) showed that M-Cdk activation depends on regulatory phosphorylation and dephosphorylation, not just cyclin concentration.

Question 13

How is the activity of Cyclin:Cdk complexes, especially M-Cdk, regulated?

Answer 13

• M-Cdk is first inhibited by phosphorylation via Wee1 kinase.
• Later, Cdc25 phosphatase removes this inhibitory phosphate, activating the Cyclin:Cdk complex and triggering mitosis.

Question 14

How is M-Cdk (MPF) activity tightly regulated during the cell cycle?

Answer 14

M-Cdk (Cyclin B + CDK1) activity is regulated by:

• Cyclin accumulation (M-cyclin), which binds to CDK1.
• Wee1 kinase, which inhibits M-Cdk by adding an inhibitory phosphate.
• Cdc25 phosphatase, which activates M-Cdk by removing the inhibitory phosphate.
  This results in a sudden surge in M-Cdk activity at the G₂/M transition, triggering entry into mitosis.

Question 15

The 3 checkpoints

Answer 15

1. G2/M: passing this point represents commitment to mitosis
2. Spindle checkpoint: ensures all of the chromosomes are attached to the spindle in preparation for anaphase.
3. START (yeast) & restriction point (animals) G1/S: cell decides whether to divide or not.

Question 16

M checkpoint control/function

Answer 16

To ensure all chromosomes are attached before anaphase

Question 17

Function of anaphase promoting complex

Answer 17

Question 18

Signalling from unattached kinetochores

Answer 18

Question 19

Anaphase-promoting complex - activation

Answer 19

Sensing system at spindle checkpoint not well understood, but involves:
Mad2
Triggered by the presence of all chromosomes at metaphase plate and tension on microtubules

APC activator active only after all chromosomes attached.

Question 20

Anaphase-Promoting Complex (APC) – Role 1

Answer 20

Activated APC marks a protein called securin for destruction by the proteasome, by addition of ubiquitin (when activated by the activating subunit)

Securin inhibits another protease called separase

The released separase destroys cohesin

Question 21

Anaphase-Promoting Complex: Role 2

Answer 21

Anaphase Promoting Complex (APC) degrades

M cyclins

S cyclins

Question 22

Reforming the Nuclear Envelope

Answer 22

In prophase, M-cyclins phosphorylate lamins, so the nuclear envelope breaks down

After M cyclins destroyed, ‘lamins’ lose their phosphate groups.

Nuclear envelope to reforms around each set of chromosomes

Question 23

cells vs oraganisms

Answer 23

Question 24

START: Starting the cell cycle

Answer 24

Cells (in animals) only replicate when the external environment sends cues to stimulate cell division / the cell cycle.

Mitogens – stimulate cell division primarily by overcoming intracellular breaking mechanisms

Growth factors - stimulate cell growth (increase in size and mass)

However, in reality, these terms are used interchangeably

Question 25

G1/S cyclins

Answer 25

Activate Cdks in late G1 Help trigger progression through start, resulting in entry to the cell cycle. Levels fall in S phase

Question 26

Starting the cell cycle: mitogens & G1/S-Cdks

Answer 26

- Mitogen binds to the mitogen receptor. - A signal pathway including MAP kinase activates G1/S-Cdk by phosphorylation - Activated G1/S-Cdk phosphorylates Rb, which releases/activates E2F transcription regulators. - The released transcription regulators activate transcription of genes that allow the cell to progress through the restriction point.

Question 27

Origins of replication and the pre-replicative complex (PreRC)

Answer 27

Eukaryotic cells have multiple origins of replication on each chromosome. The Origin of Replication complex, is attached to this at all stages of the cell cycle. A pre-replicative complex (preRC) assembles at replication origins in early G1 The preRC includes DNA helicase (unwinds DNA) Once replication starts, pre-replicative complex is destroyed, but not the origin of replication complex

Question 28

Role of S-cyclins: Entering S phase and control of DNA replication

Answer 28

S-Cdk activates DNA helicases in the prereplication complex to initiated DNA replication S-Cdk prevents assembly of new preRCs Activation of Anaphase Promoting Complex targets S cyclins for destruction during mitosis, allowing the prereplicative (PreRC) complexes reform.

Question 29

Discovery of p53

Answer 29

A virus ‘immortalised’ cells grown in the lab, giving them many of the properties of cancer cells. A single virus protein is responsible This virus protein was isolated was bound to P53 This discovery revolutionised our understanding of how cells, including cancer cells, grow and divide.

Question 30

The multiple roles of active P53 following DNA damage

Answer 30

Question 31

P53 response to DNA damage

Answer 31

With DNA damage, P53 is phosphorylated, which is active and stable. P53 is a transcription factor, and binds to the regulatory region of the p21 gene leading to transcription of P21 P21 inactivates G1/S-Cdk and S-Cdk

Question 32

P53 gene with a deleterious mutation

Answer 32

Take home: Mutations in P53 mean that the cell can continue to divide despite having DNA damage. UV-specific p53 mutations have been reported in 50% of human basal cell carcinoma (BCC) and in over 90% of squamous cell carcinoma (SCC) (the most common types of skin cancer).

Question 33

Types of genes, that when mutated can lead to cancer.

Answer 33

Proto-oncogenes: a gene which codes for proteins often involved in signal transduction from mitogens. When a mutation permanently activates the gene turn into oncogenes. e.g. E.g. MAPK Tumour suppressors: or anti-oncogene, is a gene that regulates the cells response to damage. When mutated, cells divide despite damage to DNA, E.g. Rb, p53, p21