How is stage G1 of the cell cycle defined and measured?
G1 is the "resting" phase, a gap before DNA synthesis.
- 2C DNA/cell
- General biosynthesis (growth/maintenance)
- External signals and internal state combine to make decision to remain in G1 (G0 if inactive for a long time) or divide (proceed to S)
- External signals: receptor activation
- Internal state: size, DNA integrity
- Timing can be measured using FACS to measure DNA content of cells stained with a DNA-binding dye
How is stage S of the cell cycle defined and measured?
S is the synthesis stage.
- Synthesis of histones + enzymes required for DNA replication
- Replication of DNA from multiple rep. origins
- Removal of rep. licensing factors from origin recognition complex ensures replication occurs only 1x per cycle
- Centrioles duplicate
- Timing can be measured by pulse-labeling cells in S phase with fluorescent or radioactively tagged nucleotides, or using FACS to measure DNA content of cells stained with a DNA-binding dye
How is stage G2 of the cell cycle defined and measured?
G2 is primarily a time of preparation for mitosis.
- Continued synthesis of proteins required for mitosis and cytokinesis
- Addition of cohesins links sister chromatids
- Addition of condensins and histone H1 phosphorylation condenses chromosomes at the start of M (mitosis)
- Timing can be measured as the time required for cells that were pulse-labeled during the S stage to reach mitosis
How is stage M of the cell cycle defined?
The mitosis stage:
The M stage is activated by a cell-cycle regulatory kinase.
Cells that fail to complete mitosis may become polyploid or apoptose.
What is prophase?
Prophase is the first stage of mitosis.
- Chromosomes condense
- Duplicated centrosomes separate
- Spindle forms and elongates via microtubule growth
- Kinetochores assemble onto centromeric DNA
- Nuclear envelope is intact
What is prometaphase?
Prometaphase is the second stage of mitosis, following prophase.
- Nuclear envelope breaks down (cyclin-dependent lamin phosphorylation)
- Spindle invades nucleus
- Kinetochores capture microtubules
What is metaphase?
Metaphase is the third stage of mitosis, following prometaphase.
- Bipolar attachment of chromosomes to the spindle results in equal tension from both spindle poles
- Chromosomes become aligned at the metaphase plate
- Unattached kinetochores exert checkpoint control and prevent transition to anaphase; when all kinetochores are under tension, the Anaphase Promoting Complex (APC) is activated
What is anaphase?
Anaphase is the fourth stage of mitosis, following metaphase.
- The APC triggers destruction of cohesins
- Chromatids separate and move to opposite poles, dragged by shortening kinetochore microtubules
What are the two types of separation during anaphase?
Anaphase A = chromosome-pole
Anaphase B = pole-pole
What is telophase?
Telophase is the fifth stage of mitosis, following anaphase.
- Phosphatase activity reverses the effects of the mitotic kinase on chromosome condensation
- Spindle begins to disassemble
- Nuclear envelope reforms around each set of daughter chromosomes
- Preparation for cytokinesis (usually)
What are the roles of microtubule dynamics and motor proteins in each stage of mitosis?
Prophase: MTs grow between the spindle poles; shorter astral MTs radiate in other directions; kinesin motors crosslink overlapping MTs to aid spindle elongation
Prometaphase: Polar MTs invade the nucleus and are "captured" by kinetochores
Metaphase: bipolar attachment of MTs leads to metaphase alignment
Anaphase A: MTs shorten, dragging the chromosomes toward each pole; may involve MT disassembly and kinesin-related protein that pulls (-) ends at kinetochore
Anaphase B: spindle poles separate, spindle elongates; kinesin pushes from the middle, dynein pulls from the ends
Telophase: spindle begins to disassemble
How is the timing and orientation of cytokinesis controlled?
Cytokinesis usually, but not always, follows mitosis.
The orientation of the mitotic apparatus determines the position of the contractile ring, antiparallel microfilaments associated with the cell membrane and non-muscle myosin II. Cells that fail to complete cytokinesis may become binucleate.
What are the major checkpoints controlling progression from one stage of the cell cycle to the next?
G1/S border (enter S): trigger DNA replication machinery; cyclin D + cdk4 + cdk6
G2/M border (enter M): trigger assembly of mitotic spindle; cdk1 + cyclin B
Metaphase/anaphase transition (exit M): APC turns off CDK, activates separase
Multiple Cdks are needed to regulate cell cycle progression, even beyond the Cdks mentioned here.
How are Cdks regulated?
How are Cdks activated?
Note: Protein phosphatase removal of the inhibitory phosphate starts a positive feedback loop that stimulates the activation of more Cdk molecules, making Cdk activation almost an all-or-none event.
How are Cdks inactivated?
- E3 ubiquitin ligase
- Removal of activating phosphate
What is the mechanism of the metaphase-anaphase checkpoint?
The Anaphase Promoting Complex (APC) is an E3 ubiquitin ligase involved in the transition from metaphase (aligned chromosomes) to anaphase (separated chromosomes). It does 2 things:
- Turn off the mitotic Cdk
- Activate the protease separase, which chews up the cohesin complex and promotes chromosome separation
What is the DNA damage repair pathway?
DNA damage will delay cycle progression by increasing synthesis and phosphorylation of p53. Activation of p53 increases the conc. of specific proteins such as p21, which bind to Cdks and block their activity. Once the damage has been repaired, the cell cycle continues.
Under some circumstances, activation of the p53 pathway will lead to cell death rather than cell cycle arrest.