handout 7, 5 and 6

• Signaling


  Works in a combinatorial fashion in inter-connected networks
• Cell division


  1. Mitosis

  2. Prophase

  3. Metaphase

  4. Anaphase

  5. Telophase

  6. Cytokinesis

  
• The human body experiences about 10 quadrillion cell divisions in a lifetime
• Aurora kinases


  • There are multiple Aurora kinases (A, B, C)
  • They are dysregulated in different cancers
• Polo-like kinases


  • There are multiple Polo-like kinases (1-4)
  • They are dysregulated in different cancers
• Chromosome condensation


  1. Condensin initiates chromosome compaction

  2. Cohesin forms rings that keep the chromatids together from S-phase to anaphase
• Centrosome duplication, separation and the cell cycle


  1. Duplication starts at late G1 phase, is finished by end of S-phase

  2. Controlled by phosphorylation of centrosome proteins by Cdk2 and others

  3. By late G2-phase the centrosomes 'mature', acquire PCM, become phosphorylated, helps recruit more γ-tubulin

  4. The centrosomes begin to separate from each other in late G2-phase

  5. Myosin II and cytoplasmic dynein-1 are involved

  6. Kinesin-5 and kinesin-12 are involved in pushing the centrosomes apart during mitosis

  
• If a cell needs to stop (arrest) in S-phase, it may have too many centrosomes
• If a cell loses count of centrosome number, it may have too many centrosomes
• Building the mitotic machine - the bipolar mitotic spindle


  1. Microtubules grow in a 'sunburst' arrangement during prophase

  2. Phosphorylation of proteins in the PCM by Polo-like kinase stimulates nucleation of spindle microtubules

  3. Microtubular growth and molecular motors form the mitotic spindle

  
• Chromosome alignment at the metaphase plate


  1. Kinetochores assemble at each centromere and attach each chromatid to microtubule bundles

  2. Chromosomes & microtubules must attach properly during prometaphase

  3. Kinetochore contacts the sidewall of MT & slides along MT using its motors

  4. Chromosome attached to the + end of MT bundle from one spindle pole

  5. Oriented at the center of the cell - 'congression'

  
• Metaphase is brief, at the moment that attachment/congression is finished, cells commit to anaphase
• Triggering anaphase


  1. Protein degradation by proteasome controls progression through mitosis

  2. Securin degradation triggers anaphase

  3. CyclinB begins its degradation just before anaphase, not afterward
• Chromatid movement during anaphase

  1. Anaphase A: shortening of 'chromosomal spindle' at both plus and minus ends, movement of the chromosomes toward poles

  2. Anaphase B: movement of the poles apart = elongation of the polar spindle

  3. Microtubule depolymerization powers chromatid movement
• Spindle assembly checkpoint


  Determines whether all chromosomes are properly attached and aligned at the metaphase plate
• If MAD2 is deleted using CRISPR-Cas9, it would likely result in abnormal chromosome segregation and decreased activity of APC
• Aneuploidy was the first genomic abnormality identified in cancers, and over 70% of all solid tumors show aneuploidy
• Cytokinesis


  1. MT spindle disassembles

  2. Chromosomes cluster, decondense

  3. Nuclear envelope reassembles

  4. Golgi and ER reform

  5. Contractile ring forms, contraction by myosin II splits the cell in two

  
• Cell migration


  Required for many processes in higher vertebrates like tissue/organ development, wound healing, protection against infection, and cancer metastasis
• Fibroblast movement


  1. Lamellipodium provides protrusive force through actin polymerization dynamics and treadmilling

  2. Actin-binding proteins and Arp2/3 complex nucleate F-actin and form 'Y-branches'

  3. Adhesion mediated by proteins like vinculin and integrins

  4. Pulling near the front and pushing at the rear require myosin II
• Listeria monocytogenes uses F-actin polymerization for its movement and infection
• Cytochalasin, an inhibitor of actin polymerization, prevents the spread of Listeria infection from one cell to its neighbors
• Tin polymerization


  The process of forming polymers from tin monomers
• Actin 'tail' formation


  1. Filament polymerization on the bacteria surface

  2. Begins to 'push' the bacteria

  3. Tail length is maintained via treadmilling
• Listeria infection could not be spread from one cell to its neighbors on a petri dish in the presence of cytochalasin (an inhibitor of actin polymerization)
• Cell death


  The process by which a cell ceases to function and eventually dies
• Apoptosis


  • Proteins are specifically degraded
  • Cell shape changes (overall shrinkage in cell volume)
  • Membrane blebbing
  • Cell detachment
  • DNA fragmentation
• Necrosis


  The swelling of both the cell and its internal membranous organelles, membrane breakdown, leakage of cell contents into the extracellular space, resulting in inflammation
• Around 330 billion cells are replaced daily in our bodies. Over about 3 months, this equals the number of cells in our entire body
• Examples of apoptosis during development


  • Interdigital apoptosis to transform a limb bud into a real foot/hand
  • Apoptosis used to create many hollow structures in our bodies; ducts in breast
  • The tadpole loses its tail to become a toad by apoptosis
  • Males lose their female side and females lose their male side by apoptosis
• Caspases


  Proteases that cleave many proteins involved in apoptosis
• Caspases are present in every cell as inactive enzymes called pro-caspases
• Types of caspases


  • Initiator caspases: 2, 8, 9, 10
  • Executioner caspases: 3, 6, 7
• A peptide 'drug' called zVAD-fmk inhibits all caspases, many other selective caspase inhibitors also exist
• Apoptosis appears to be involved in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's
• In type 1 diabetes, cells are abnormally killed via apoptosis
• In cancers, damaged cells evade and escape the ability to die, allowing their evolution
• Extrinsic apoptosis pathway


  Uses 'death' ligands and 'death' receptors
• Apoptosis during development appears to use both the extrinsic and intrinsic pathways
• Intrinsic apoptosis pathway


  • Mitochondrial outer membrane permeabilization (MOMP) is coupled to cell death
  • MOMP occurs 'all at once' and very fast
  • MOMP is caspase-independent (zVAD-fmk)