Control of Cell Division
G1, S, G2, M
- Very early experiments just showed mitosis – wait – mitosis – wait, etc
- Later exps showed mitosis – wait – DNA replication – wait – mitosis – etc.
o everything in between was just a gap
- human cells
o G1 – 9 hrs
o S – 10 hrs
o G2 – 4.5 hrs
o M – 30 min
- G0 – cell that is not actively progressing through cycle is in G0 (like neurons)
- Mitosis
o Prophase – chromosome condensation and nuclear breakdown
§ Each chromosome consists of two sister chromatids at this point
o Metaphase – lining up
o Anaphase – separating
o Telophase – nuclear rebuild and chromosome condensation
o Cytokinesis
- Cell cycle takes 24hr in human cells, but as little as 90 min in S. cerevisiae
Studying cell cycle with genetics in yeast
- Saccharomyces yeast multiply by budding
o Con correlate position in the cell cycle by looking at budding
o Mutagenize cells and look for mutants in cell cycle
§ Problem – if can’t go through cell cycle then they don’t multiply!
§ Solution – temperature sensitive mutations
§ Weinert and Hartwell did a big screen for these
§ Grow at permissive temperature, switch to restrictive temp and look for cells arrested all at the same phase of the cell cycle (Fig. 19.3)
§ Nobel prize…..
CDC28 and cyclin/cdks
- CDC28 one mutant identified in screen above
- This is the commitment step of the cell cycle
o If CDC28 is wt, cell is committed to starting the cell cycle
o If CDC28 is mutant, cell does not start new cell cycle
- CDC28 was identified and found to be a protein kinase
o Found homologous protein in other organisms like Xenopus (frog)
o Homologous proteins could substitute for each other in other organisms, so have same function
General principles of cell cycle progression
- heterodimeric kinases drive cell cycle
- consist of
o catalytic subunit – cyclin-dependent kinase (cdk)
o regulatory subunit – cyclin
§ cyclins are produced (transcribed and translated) for only periods of the cell cycle
§ at the end of this phase of the cell cycle, the cyclins for that phase are completely degraded
· this regulated degradation leads cell cycle to go only one way
- may be one cdk and different cyclins (yeasts)
- or different cdk/cyclin pair for each phase (humans)
o in each phase, they phosphorylate different proteins, this phosphorylation changes the activity of these proteins and drives the cell cycle
What are some targets of cyclin/cdks, and how do they push the cell cycle?
- at beginning of S phase, phosphorylates proteins involved in initiating DNA replication
- in prophase of mitosis, phosphorylates proteins needed to condense chromosomes
- in prometaphase, phosphorylates lamins, proteins that maintain the nuclear membrane
o when phosphorylated the lamins become soluble and nuclear membrane is broken up
How cyclin/cdks control the G1-to-S transition
- a transcriptional activator protein called E2F increases transcription of a number of genes for proteins involved in DNA synthesis
- this activator protein must only be active at the end of G1 and through S phase
- How?
o E2F is always around, but early in G1 is bound to the inhibitor Rb protein, so it is inactive
- Need to get Rb off of E2F
- CyclinD/cdk4 and cyclinE/cdk2 are crucial for this
- They phosphorylate Rb, making it fall off E2F
- Now E2F induces txn of DNA replication genes
o Including cyclinA, which complexes with cdk2 and this cyclin/cdk phophorylates Rb, keeping E2F active
Rb is called a tumor suppressor
- if no Rb, then E2F active all the time
- constantly pushing cells into S phase, even when inappropriate
- too much cell cycle – cancer
- mutations of Rb lead to retinoblastoma cancer