UnitE 3

• What is required for M Phase

  • Mitotic cyclins (M-cyclins) levels must increase
  • Cohesin rings must be cleaved during metaphase-to-anaphase & condensin rings must aid in chromosome condensation
  • Centrosomes must duplicate/mitotic spindle must assemble

  
• Mitotic cyclins (M-cyclins)


  Cdk complexes promote cell entry into mitosis

  
• 1. cdk complex activation


  1. Active M-cdk complex acts as positive feedback to phosphorylate more Cdc25 which in turn activates more M-Cdk

  2. Active M-Cdk phosphorylates and inactivates Wee1 kinase

  3. Leads to higher concentration of active M-Cdk

  4. Active M-Cdk will also activate APC/C which targets mitotic cyclins (i.e Cyclin B) for degradation towards the end of mitosis

  
• Cohesin rings


  • Keep sister chromatid together prior to metaphase
• Condensins


  • Rings responsible for reorganization and condensation of chromosomes during early mitosis

  
• Cohesin and condensin assembly

  Assemble on DNA once activated by M-Cdk
• Cohesin and condensin


  • Both are responsible for preparing the chromosomes prior to sister chromatid separation
• Centrosome duplication


  • Centrosomes must duplicate themselves during S/G2 transition
• Mitotic spindle assembly


  1. Mitotic spindle assembly depends on properties of microtubules

  2. Once centrosomes have duplicated and cells enter mitosis, they separate and nucleate array of microtubules called asters

  3. The two centrosomes then move to opposite ends of the nucleus to form spindle poles

  4. Asters help hold two centrioles at opposite poles and help maintain structure of the mitotic spindle

  
• Microtubules


  • Ability to polymerize and shrink by addition or loss of tubulin subunits (dynamic instability)
• 1. Cdk phosphorylation of microtubule-associated proteins

  Increases dynamic instability of microtubules at start of M phase

  
• Chromosome attachment to mitotic spindle

  1. Mitotic spindle assembly begins at prophase and microtubule attachment to the kinetochore takes place during prometaphase, following nuclear envelope degradation

  2. Inner and outer kinetochores proteins assemble onto the centromere locus

  3. Binding of centromere proteins allows for recruitment of outer kinetochore proteins that in turn bind microtubules

  
• Chromosome alignment


  1. Once chromosomes have attached to their respective microtubules, they begin aligning along equatorial plane of the mitotic spindle

  2. This alignment and positioning defines start of metaphase

  3. Chromosomes are continuously in motion due to the dynamic instability of microtubules

  
• Sister-chromatid separation at Anaphase

  1. Anaphase is initiated by the degradation of cohesin rings and physical separation of chromosomes

  2. Cohesin is cleaved by systein protease called separase

  3. Prior to anaphase, separase is bound to and held inactive by securin

  4. At onset of anaphase, APC/C will target securin for degradation, releasing separase

  5. Separase in turn will cleave the cohesin subunit (Scc1 in yeast)

  
• Anaphase can only happen when all chromosomes are attached to microtubules (MTs) and properly aligned at the metaphase plate, otherwise, daughter cells will not have the appropriate number of chromosomes

  
• Spindle assembly checkpoint (SAC)

  • Monitors that all chromosomes are attached to microtubules and properly aligned
  • APC/C will target Cdc20 for degradation, allowing anaphase to take place
  • APC/C will target M cyclin (Cyclin B) for degradation, thus inactivating mitotic Cdk

  
• Activation of APC/C


  1. Mad2 can exist in either a closed or open form (cMad2 and oMad2, respectively)

  2. In the closed form (cMad2) it can bind to and inhibit Cdc20

  3. Mad2 is converted into the closed form by associating with kinetochore-bound Mad1

  4. A single unattached kinetochore is sufficient to produce enough cMad2 to inhibit all of the cellular Cdc20 and prevent anaphase

  5. When all kinetochores are properly attached to microtubules: Mad1 dissociates from the kinetochore and stops producing cMad2

  6. A protein called p31comet is then able to displace cMad2 from Cdc20

  7. The free Cdc20 can then associate with APC/C to produce active APC/C

  
• Only bi-oriented chromosomes inactivate the SAC
• Microtubule attachment to kinetochores

  • Amphitelic – both kinetochores are attached to MTs from the two spindle poles
  • Merotelic – only one kinetochore is attached to MTs that come from both spindle poles
  • Syntelic – Both kinetochores are attached to MTs coming from one spindle pole
  • Monotelic – only one kinetochore is attached to MTs

  
• Nuclear envelope reformation during Telophase

  1. Mitotic spindle disassembles and nuclear envelope reforms around both clusters of chromosomes

  2. Dephosphorylation of nuclear lamin proteins allows them to reassemble and build a new nuclear envelope
• Cytokinesis


  1. The cytoplasm is divided in two by the contractile ring. The closure of the cleavage furrow is aided by non-muscle myosin II

  2. Other cytosolic components such as the cytoskeleton, organelles and other cytosolic proteins are equally distributed between the newly-formed daughter cells

  3. Chromosomes are fully decondensed and the nuclear envelope is reformed

  4. Microtubules emerge from the single centrosome in each daughter cell

  5. The remaining microtubules from the mitotic spindle that are no longer attached to are referred to as the midbody

  
• Cell size regulation


  Cell growth, cell division, and cell death
• Mitogens


  Stimulate cell division

  
• Growth Factors (GFs)


  Stimulate cell growth
• Survival Factors


  Promote cell survival by inactivating apoptotic mechanisms
• Mitogen-induced cell division


  1. Mitogen (extracellular signal) binds to its receptor on cell surface and induces a signaling cascade of events

  2. This leads to activation of G1-Cdk and G1/S-Cdks

  3. Rb (retinoblastoma) protein binds and inactivates transcription of cell cycle proliferation proteins

  4. When G1-Cdk and G1/S-Cdks are activated, they phosphorylate Rb = inactivating the protein

  5. This results in activation of transcription of S-phase specific genes = cell proliferation

  
• Growth factor-induced cell growth

  1. Extracellular growth factor (i.e hormone) binds to its receptor

  2. This activates downstream cascade of signaling events that result in activation of Tor

  3. Tor, in turn promotes cell growth via several mechanisms: inhibiting protein degradation processes, activating protein synthesis

  
• Apoptosis


  • Cell death mediated by a family of proteases called caspases
  • Two types: initiator and executioner caspases
  • Initiator caspases cleave and activate downstream executioner caspases which in turn disassemble proteins

  
• Bcl2 family of proteins

  • Bax and Bak promote cell death by inducing release of ETC protein cytochrome c from mitochondria to cytosol
  • This release of cytochrome c activates initiator caspases and promote assembly of apoptosome
  • Apoptosome recruits Caspase-9 and promotes signaling cascade for apoptosis

  
• Spindle Assembly Checkpoint (SAC)

  A mechanism that ensures accurate chromosome segregation during cell division by preventing the onset of anaphase until all chromosomes have attached to the spindle apparatus and aligned at the metaphase plate
• RB (Retinoblastoma) protein


  A tumor suppressor protein that regulates the cell cycle by inhibiting the transcription of genes required for cell cycle progression
• APC/C


  An E3 ubiquitin ligase that plays a critical role in the regulation of the cell cycle, including the onset of anaphase
• Securin


  A protein that is targeted for degradation by APC/C at the onset of anaphase, which allows sister chromatids to be separated
• Cyclin B


  A protein that is targeted for degradation by APC/C at the onset of anaphase, which allows the cell to exit mitosis