Lecture 19: BICD 110 FINAL

Eukaryotic cell cycle

the cell cycle is the foundation of?

development and homeostasis

homeostasis x cell cycle

• adaptive immune responses (T cells)

• small intestine lining regeneration every couple of days

• epidermis turnover

main goal of the eukaryotic cell cycle

duplicate DNA and equally partition chromosomes

how many chromosomes do humans have?

46 chromosomes (22 autosomes (pairs) and 1 pair of sex chromo)

diploid cells are

2n = 2 copies of each chromosome (one maternal and one paternal)

transient state (chromosomes)

4n = 4 copies of each chromosome during replication

each daughter cell gets 2 copies (one maternal one paternal)

What are the stages of the cell cycle?

1. Interphase: G1, S, G2

2. Mitosis (M): chromosome segregation, division

3. after M: cells can re-enter into G1 or exit via G0

What is quiescence/post-mitotic state?

cells exit the cycle (G0)

the state that most cells in the human body are in

G1 (11hrs)

• cell grows in size via GEFs

• phase is the most maleable in length/time spent

• time spent in this phase depends on GFs, external cues

S phase (8 hrs)

- NO RETURN: Once a cell enters S, it is committed to the rest of the cell cycle

• DNA and chromosome replication

• Centriole duplication

G2 (4hrs)

• cell corrects DNA replication errors

• cells prepares for M phase

M Phase (1 hr)

• chromosomes segregate

• cell divides and separates

Temperature sensitive alleles - Yeast

• traditional model of cell cycles

• S cerevisiae

• S pombe

• genetic screens for cell cycle defects

Frogs (oocytes)

• study biochemistry of cell cycle regulation

Cultured Mammalian Cells

• traditional cell cycle model

• cell cycle imaging of mitosis and cancer

Flow Cytometry

• studying cell cycle stages and progression based on quantifying DNA content w/i cell

1. Cells stained with fluorescent dye: propidium iodine

2. more DNA in cell = more iodine staining

3. cells put into flow cytometer

4. measures the fraction of cells with certain DNA content

*Peaks represent # cells with certain DNA content

First peak = unreplicated (G1)

first trough = undergoing replication (S)

second peak = replicated (G2)

Checkpoints

cells commit to next stage, point of no return

CDKs

• Ser/Thre Cyclin dependent kinases

  • phosphorylate 100s targets to drive next cell cycle stage

• primary driver of cell cycle activity

• low kinase activity when unbound by cyclins

• Levels remain CONSTANT at all times, only ACTIVITY changes

Cyclins

• small proteins that bind and activate CDKs kinase activity

• levels CHANGE depending on cell cycle

• prime initiation of next cycle stage, ensuring UNIDIRECTIONAL movement

Cyclin/CDK for G1

Cyclin D, CDK 4

Cyclin/CDK for G1/S

Cyclin E, CDK 2

Cyclin/CDK for S

Cyclin A, CDK 2

Cyclin/CDK for M

Cyclin B, CDK 1

Cell Cycle Progression: G1 phase

1. EC growth signals bind RTKs

2. MAPK phosphorylated and downstream cascade activated

3. Downstream TFs transcribe cyclin D

4. Cyclin D binds and activates CDK 4

Cell Cycle Progression: G1 to S phase

1. Cyclin D/CDK4 phosphorylates and inhibits Rb protein

2. Rb translocates out of nucleus

3. E2F TF is now active and able to transcribe cyclin E

4. cyclin E/CDK2 phosphorylates Rb, leading to increasing levels of this complex (POSITIVE FEEDBACK)

5. eventually, levels are high enough to trigger progression to S phase

Cell Cycle Progression: S phase

1. spike in cyclin A/CDK2 activity leads to chromosome duplication

2. sister chromatids are now linked via cohesin

3. centrioles are dublicated

Cell Cycle Progression: S to M phase

1. Wee 1 kinase normally phosphorylates & inactivates CDK1

2. CDC25 removes inhibitory p from CDK1, activating it

3. CyclinB/CDK1 complex levels rise, phosphorylating & activating CDC25 (POSITIVE) and inhibiting Wee1 kinase (NEGATIVE)

4. complex goes on to p 100s targets, pushing cell to M phase