slide set 16 Flashcards
cell divisions in your body
bone marrow stem cells: 1,000,000/min
cell theory
all cells are made up of other cells
cell cycle basics
Cell must
- grow
- replicate its genome
- centrosomes must be segregated to opposite ends of the still-growing cell
- cytoplasm is cleaved in half
- 2 daughter cells, identical in size to original cell
S+M phase
S Phase: chromosomes are duplicated (entire genome is duplicated)
M Phase: Mitosis and Cytokinesis
4 cell cycle stages
Interphase
- M phase
- G1 phase
- S phase
- G2 phase
G = gap
mitosis is easy to see through a microscope, but what about the other stages
- we can detect S phase
How can we identify S phase?
- detect cells that are replicating their DNA
- 2 options
- Detect S-phase by DNA replication and incorporation of a modified base
* detect BrdU with an antibody
* treat cells with BrdU for a certain amount of time- if it is dividing, it’ll include BrdU
- if it isn’t, the cell won’t take in any bases floating around
* bind antibody to BrdU, cell that fluoresces has been in S phase
- Detect S-phase by DNA replication and incorporation of a modified base
- Measure relative DNA content per cell by flow cytometry = fluorescent label on DNA is detected
How can we identify S phase (part 2)?
harvest cells
isolate cells
stain cells
- Measure relative DNA content per cell by flow cytometry = fluorescent label on DNA is detected
fluorescent dye shows us how much DNA is there
height of peak = # of DNA in cells
peaks change bc cells divide at random times
cell cycle controllers
- proteins control when each process occurs
- These proteins integrate signals and can slow or stop the cell cycle if conditions are unfavorable or if errors have occurred
CDK aka
cyclin-dependent kinase
key enzyme family
Cdk’s
require a cyclin partner before they are active as a kinase
major Cdk’s and their cyclin partners
each active Cdk recognizes and phosphorylates specific target proteins that ultimately drive a cell cycle phase
focus on M-Cdk (mitotic Cdk)
Cdk enzyme activity over course of cell cycle stages
Cdk enzyme activity rises abruptly and declines abruptly at cell cycle stages
what happens if we add an inhibitor that blocks cells from getting to S phase
we’d have one really large peak in G1
Rise in Cdk activity is based on…
synthesis and degradation of cyclins
M phase questions
Are all chromosomes attached to the spindle?
(If not, we may get uneven daughter cells)
Metaphase to Anaphase transition
G1
restriction point: point of no return, after cell crosses that point cell is committed to rest of cell cycle and dividing
Is environment favorable?
enter cell cycle! proceed to S phase!
G2 questions
Is all DNA replicated?
Is environment favorable?
G2/M transition!
Structural basis of Cdk activation and additional regulators
- inactive state: T loop is folded (part of active site of kinase)
- kinase can’t interact with target protein
- without cyclin bound, active site is blocked by region of T loop
- As cyclin is made, cyclin protein can bind to CDK
- binding of cyclin causes T loop to open up
- active site of kinase is revealed!
- CAK adds phosphate to CDK (now it is fully active)
To be active, CDK must
- bind to cyclin
- be phosphorylated by CAK
Cdk’s are regulated by inhibition of enzyme activity
- Wee1 kinase can come in and phosphorylate a different part of the active Cdk-cyclin and inhibit the activity
- Cdc25 phosphatase can remove this inhibitory phosphate group
M-Cdk partners
positive feedback loop
produces rapid rise in active Cdk1 to drive mitotic entry
- Cdk1 binds to M-cyclin as M-cyclin levels rise
- M-Cdk is phosphorylated on active site by CAK and on a pair of inhibitory sites by Wee1 kinase
- leads to inactive M-Cdk complex
- inactive M-Cdk is activated at end of G2 by Cdc25 (a phosphatase)
- Cdc25 is further stimulated by active M-Cdk, results in positive feedback
- feedback is enhanced by ability of M-Cdk to inhibit Wee1
Inhibitors used at G1/S and S phases
CKI’s (cyclin-dependent kinase inhibitors)
p27 distorts the kinase active site and inactivates enzyme activity
When a process is complete,
enzyme activities rapidly fall when a process is complete
cyclins are destroyed by proteolysis
proteolysis: protein is no longer needed
Polyubiquitin chains
target a protein for degradation by the proteasome
requires an E3 ubiquitin ligase
proteolysis example
mitotic exit is controlled by proteolysis through APC/C (anaphase promoting complex)
transition from G1 to S phase
G1 to S phase through degradation of CKI’s
environmental cues
cell cycle machinery receives environmental info to slow timing as needed
How to get G1 going
Need active E2F, a transcription factor for expression of S-Cdk, enzymes and proteins for DNA replication
this will drive the cell from G1 to S
How is E2F responsive to cues from the environment?
- Mitogen binds receptor, activates Ras
- Ras activates MAP kinase cascade
- MAP kinase cascade leads to activation of a transcription factor that leads to expression of Myc and cyclin
- Active G1 Cdk phosphorylates Rb, a protein that inhibits E2F
resulting G1/S-Cdk and S-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop
E2F proteins also stimulate transcription of their own genes, another positive feedback loop
starting S phase
- Mitogen binds receptor, which activates Ras
- Ras activates MAP kinase cascade
- MAP kinase cascade leads to activation of a transcription factor that leads to expression of Myc and cyclin
- Active G1 Cdk phosphorylates Rb, a protein that inhibits E2F
positive feedback at end leads to more E2F
Rb
Retinoblastoma protein
active when not phosphorylated
How can we slow/stop the process so damage can be repaired?
*
p53
p53 prevents cell cycle from proceeding until DNA damage is repaired
p21 binds to active S-Cdk and inactivates it (now S phase cannot continue)
