Cytoskeleton 1

Question 1

Intermediate filament structure

Answer 1

1. Basic structure is two-chained coiled coils that co-translationally become dimers and are held together by hydrophobic interactions.
2. Coiled coils assemble in antiparallel fashion to form tetramer.
3. Tetramers form higher order assemblies, 10 nm filaments.
4. N-terminal and C-terminal ends are globular; coiled coil region interrupted by linker domains.
5. Intermediate filament as a whole are not polar even though individual coiled coil domains are polar.

Types of intermediate filaments and disease associations

1. Lamins: mutations lead to laminopathies
2. Keratins:mutations lead to skin blistering diseases

Question 2

Properties of Actin

Answer 2

• Monomer is G-actin
  
  • Has a + and - end
  • Binds to ATP or ADP
• Polymer is F-actin
• Has polarity due to actin filament having two ends that are different.
  
  • Plus end is bound to ATP
  • Minus end is bound to ADP
• Polarity allows motors to move in particular direction

Question 3

Polymerization of actin

Answer 3

1. Rate limiting step: Formation of nucleus which requires 3 G-actin monomers
2. After nucleation, you have rapid growth which occurs faster at the + end or barbed end where G-actin bound to ATP is being added.
3. The G-actin-GTP is being added to plus end faster than hydrolysis of G-actin-GTP to G-actin-GDP in the minus end

Question 4

Properties of Microtubules

Answer 4

• Monomer is tubulin which is a heterodimer made of alpha-beta tubulin
• • end has B-tubulin at edge and is GTP bound
• • end has A-tubulin at edge and is GDP bound

Question 5

Polymerization of Microtubules

Answer 5

1. Beta-alpha tubulin heterodimer assembles into a protofilament with a Beta-tubulin + end and a alpha-tubulin at - end
2. 13 protofilaments are then arranged around gamma-tubulin ring complex to form a microtubule
   
   1. Gamma tubulin ring is at the - end

During protofilament assembly, GTP-tubulin is added to + end faster than hydrolysis of GTP to GDP-tubulin.

*Adding GTP-tubulin cap stabilizes + end

*GDP-tubulin units in the + end destablize microtubule causing depolymerization.

Question 6

Motile vs. Nonmotile cilia

Answer 6

1. Non-motile cilia
   
   1. Are involved in signaling during development
   2. House Shh pathway
2. Motile cilia
   
   1. Found in cilia and flagella
   2. Have central pair of microtubules unlike non-motile cilia
   3. Have knobs between microtubules unlike non-motile cilia

Question 7

Centrosome

Answer 7

1. Microtubule organizing center
2. Microtubules eminate from centriole such that - end is capped at the centriole and + end can extend out towards cell periphery and nucleus.

Question 8

Three different types of microtubules

Answer 8

1. Astral MTs- can carry things to cell membrane
2. Kinetochore MTs – have + ends that are attached to kinetochore allowing chromosome to be caught and aligned on metaphase plate
3. Interpolar MTs-have + ends interacting with other MTs coming from the other centriole.

Question 9

Microtubule Associated Proteins (MAPs)

Answer 9

1. Stabilize + end or - end
2. Bind to side and stabilize side binding or bundle formation
   
   1. Tau binds to side of MTs and forms crosslinks thereby stabilizing MTs
   2. Defective Taus implicated in neurofibrillary tangles in AD.

Question 10

Microtubule + tip proteins

Answer 10

1. For microtubules to reach cell periphery, + tip proteins bind to + tip to prevent catastrophe and to allow MT to grow
2. • tip proteins are important for
     1. Communication and connection with cell cortex and periphery
     2. Motors associate with + tips to carry proteins or cargo
     3. Capture chromosomes during mitosis by associating with kinetochore

Question 11

Toxins of actin filaments

Answer 11

1. Phallodium
   
   1. Binds and stabilizes actin filaments thereby preventing them from being dynamic
2. Alpha-amanitin
   
   1. RNA pol II inhibitor

Question 12

Microtubule Toxins

Answer 12

1. Colchcine- depolymerizes microtubules
2. Taxol- binds and stabilizes microtubules thereby impacting microtubule dynamics in mitotic spindle apparatus
   
   1. Stabilizing mts prevent them from moving back towards centrosome after capturing chromosomes

Question 13

When and how do cells migrate

Answer 13

1. When
   
   1. During development: path finding and targeting of neurons
   2. Chemotaxis: migration of neutrophiles to sites of infection
   3. Cancer: metastasis
2. Cell migration and intracellular transport can be:
   
   1. Motor driven: motors like dynein, myosin, kinesin
   2. Polymerization driven
3. How does cell migration occur:
   
   1. Can use motile cilia and flagella OR
   2. Actin polymerization
      
      1. Actin polymerization at the leading edge of membrane and myosin II dependent contraction at the - end allow cell to move.
      2. Process
         
         1. Ligand binds to transmembrane protein
         2. Rho GTPases are activated
         3. Rho activates WASP/SCAR
         4. WASP/SCAR activate Arp 2/3
         5. Arp 2/3 promotes nucleation on the side of actin filaments in order to promote branching of actin fibers near cell membrane at leading edge of lamellipodia such that they push against the cell and cause it to move.

Question 14

Listeria

Answer 14

1. Bacteria can hijack Arp 2/3 in epithelial cells
2. Bacteria expresses WASP/SCAR-like protein near cell membrane
3. Recruits Arp 2/3
4. Arp 2/3 promotes polymerization of actin in the leading edge allowing bacteria to move.