Section 6: Mitosis and cell cycle control

Question 1

Briefly describe the 4 stages of the cell cycle

Answer 1

1. G1 (gap1) phase
   
   • Growth and metabolism
     
     • Preparing machinery for mitosis
   • Variable length depending on cell type
   • The stage where most cells are arrested when not dividing
     
     • If they are arrested, they are in ‘G0’
2. S (synthesis) phase
   
   • DNA replication
3. G2 (gap2) phase
   
   • Prepare chromosomes for segregation
   • G1, S, G2 = interphase
4. M (mitotic) phase
   
   • Chromatin condensation
   • Nuclear envelope breakdown
   • Sister chromatids attach to mitotic spindles
   • Chromatids are segregated; they decondense and reform nuclei
   • Cytokinesis

Question 2

What happens if something goes wrong in mitosis?

How do cells appear when they are undergoing mitosis?

Answer 2

• Checkpoints (after each PMAT phase) ensure chromosomes are segregated properly
  
  • If something goes wrong, the cell aborts and undergoes apoptosis
• Cells appear more rounded (due to cytoskeletal changes)

Question 3

Describe 3 characteristics of budding yeast S. cerevisiae

Answer 3

• Replicates by forming a bud
• Lacks a G2 phase, longer G1
• Nuclear membrane is never degraded

Question 4

Describe 3 characteristics of fission yeast S. pombe

Answer 4

• Grow by elongation of ends
• Longer G2 and M phase
• ‘Fission’ yeast as it has a cell wall
  
  • Septum

Question 5

What are ‘CDC’?

Answer 5

• Temperature sensitive mutants exist in S. cerevisiae and S. pombe
• They cause defects in proteins (called CDC) required to progress through the cell cycle
  
  • CDC are cyclin dependent kinases
• They are highly conserved in eukaryotes

Question 6

State the 3 protein families involved in regulating the cell cycle

Provide common examples

Answer 6

1. Kinase (ex., CDC2, CAK, Wee1)
   
   • Add phosphate
2. Phosphatase (ex., CDC25)
   
   • Remove phosphate
3. Cyclin (ex., mitotic cyclin)
   
   • Vary in concentration

Question 7

What are kinases (ex., CDC2)?

Answer 7

• Kinases depend on cyclin activity
• Kinases are always present
  
  • CDC2 is involved in M phase entry
  • Thus, it is only active at G2-M transition, when mitotic cyclin is high
• Cyclin binds to CDC2 to create MPF (mitosis promoting factor, heterodimer)

Question 8

What happens when CDC2/CDC28 is mutated?

Answer 8

• CDC2 is required for entry into M phase
• If the cell cannot enter M phase, it will not divide and thus will be larger than the wild type

Question 9

State the 4 classes of cyclin

How are cyclin regulated, since they must vary in concentration?

Answer 9

1. G1
2. G1/S
3. S phase
4. Mitotic

Regulation of cyclins is done by ubiquitin-mediated proteasome-dependent degradation

• As with any protein, they are also regulated at the transcriptional level

Question 10

What is ‘Ubiquitin’ (Ub)?

How does it degrade proteins such as cyclins?

Answer 10

• 76 aa protein found in all eukaryotic cells
• Sequential addition of polyUb (polyubiquitination) to the target protein shuttles it to proteosomes where it is degraded
  
  • Requires ubiquitin ligase (2 types: APC/C and SCF)
    
    1. APC/C helps degrade mitotic cyclin
    2. SCF helps degrade S phase cyclin

Question 11

How is MPF regulated?

Answer 11

• Since MPF triggers M phase, we only want it to be active when there is enough MPF to be successful

1. Initially, any MPF made is inactive
   
   • Wee1 inactivates it through phosphorylation
     
     • Y15 site
     • Inhibitory kinase
2. Later, MPF is needed
   
   • CAK (CDK-activating kinase) activates it through phosphorylation
     
     • T161 site
     • Activating kinase
3. MPF is still inactivated; remove the breaks!
   
   • CDC25 activates it through dephosphorylation
     
     • Y15 site
     • Activating phosphatase

Question 12

What happens when there is:

• A deficit of CDC25 or excess of Wee1
• A deficit of Wee1 of excess of CDC25

Answer 12

Question 13

What sorts of events does MPF trigger and how?

Answer 13

• Triggers events that trigger mitosis through phosphorylation (it is a kinase):
  
  • Chromosome condensation
  • Disassembly of nuclear envelope
  • Interphase microtubule disassembly and mitotic spindle formation
  • Remodeling golgi, ER

Question 14

When is active MPF inactivated during the M phase?

Answer 14

• At anaphase
• The mitotic cyclin is degraded via polyubiquitination
  
  • Uses ligase APC/C (anaphase-promoting complex)
• Mitotic cyclin contains a destruction box of amino acids that the Ub is added to

Question 15

Explain how APC/C is regulated

Answer 15

• APC/C is inactive in the presence of G1/S CDKs
• APC/C is active in the presence of CDC14

Question 16

Describe how S phase begins

Answer 16

• S phase cyclin binds to CDK
  
  • They are inhibited by an inhibitor during G1
• When the cell is ready to transition, the inhibitor is targeted for degradation by ubiquitin ligase SCF
  
  • ​SCF also directs degradation of G1 and S phase cyclin to allow mitotic cyclin increase