Cytoskeleton 2

Question 1

Actin and Myosin II functions

Answer 1

1. Cell Migration
   
   1. This is a fibroblast moving along substrate
   2. Lamellipodium is at leading edge of cell pioneering movement driven by branched actin filaments (nucleated by Arp2/3)
   3. After protrusion happens, lamellipodium will attach to substrate via focal contacts, which provide traction for cell to move along.
   4. The lagging end or tail is contracting via a myosin II contraction
2. Formation of contractile ring in cytokinesis to cleave cell into two
3. Formation of epithelial tube
   
   1. Epithelial sheet invaginates due to adhesion belt with associated actin filaments contracting in a myosin II dependent manner

Question 2

Arrangement of intermediate filaments, microtubules, and actin in neurons

Answer 2

• There are neurofilaments (type of intermediate filament)
• There are microtubules along axon
• There are actin filaments near the growth cone

Question 3

Properties and Structure of Myosin from N–> C terminus

Answer 3

1. Properties
   
   1. Binds actin
   2. +end directed
   3. Involved in cytokinesis, intracellular transport, endocytosis, etc
2. Structure
   
   1. N terminus –> C-terminus
      
      1. Head –> light chain –> neck/hinge region –> tail
      2. Head or motor domain (binds to actin)
         
         1. Has ATPase activity
      3. Light chain
         
         1. Many myosins have calmodulin in this region
      4. Neck region
         
         1. important in mechanical movement
      5. Tail (associates with cargo)
         
         1. Made of coiled coil that holds two heads together

Question 4

Process of Myosin movement along actin

Answer 4

1. Myosin is bound to ADP and is tightly associated with actin
2. Myosin’s ADP is exchanged for ATP
3. Myosin bound ATP causes its release from actin
4. Myosin undergoes a conformational change in the neck region to go up
5. ATP is hydrolyzed to ADP and Pi
6. Myosin with ADP and Pi can weakly associate with actin
7. This binding to actin accelerates Pi release such that myosin with ADP binds tightly to actin and undergoes a conformational change that brings about a power stroke

Question 5

Properties and Structure of Kinesin from N–> C terminus

Answer 5

1. Properties
   
   1. Bind microtubules
   2. Mostly + end directed
2. Structure
   
   1. From N terminus to C-terminus
      
      1. Head –> neck region –> stalk region –> light chains –> tail region
      2. Head or motor domain (associates with microtubules)
         
         1. Has the ATPase activity
         2. If head is at the N-terminus, kinesin is + end directed
         3. If head is at the C-terminus, kinesin is – end directed
      3. Neck region
         
         1. Determines polarity
      4. Stalk region
         
         1. Coiled coil allows dimerization
      5. Tail region
         
         1. Binds to various target organelles/vesicels
         2. Binds to other proteins part of kinesin
         3. Light chains are bound to tail region unline myosin with light chains bound to head.

Question 6

Process of Kinesin movement along actin

Answer 6

1. i.Kinesin/ADP on trailing head is weakly bound to MT
2. ATP binds to leading head on Kinesin
3. Trailing head rotates to move in front of leading head.
4. New trailing head hydrolyzes ATP
5. New leading head binds MT and dissociates ADP
6. Pi dissociates from new trailing head weakening heads binding to MT
7. Kinesin/ADP on trailing head is restored

Question 7

Properties and structure of dynein

Answer 7

1. Properties
   
   1. – end directed
   2. Has 6 ATPase Associated with diverse cellular Activities (AAA protein) domains
   3. Found in ciliary arms necessary for cilia and flagella beating
   4. Functions during mitosis, transport of mRNA, vesicles, organelles, and viruses
2. Structure
   
   1. Stalk–> 6 AAA domains in motorhead –> tail
      
      1. Stalk
         
         1. Binds to Microtubule
      2. AAA domains
         
         1. Have ATPase activity
         2. Energy attained from hydrolysis
         3. Conformational change
         4. Movement
      3. Tail
         
         1. Can bind to cargo
         2. Can also bind to other MT

Question 8

Kartagener Syndrome or Primary Ciliary Dyskinesia (PCD)

Answer 8

1. Cause
   
   1. Mutation in the heavy chain of dynein that is found in outer arms of cilia
2. Effect
   
   1. Dynein outer arms in cilia are missing
   2. Cilia are immotile
3. Symptoms
   
   1. Respiratory tract infections
   2. Male sterility due to lack of effective sperm movement
4. Pathology
   
   1. Immunofluorescence stain shows that in normal patient, dynein and tubulin in MTs are co-localized.
   2. In PCD patient, tubulin is malformed and dynein is not co-localized.
   3. Therefore dynein not found in cilia
   4. Dynein is still found in basal bodies and centrosome
   5. Associated with situs inversus

Question 9

Motor cargo relationships are specific

Answer 9

1. Motor tail region can directly bind to cargo OR
2. Motor tail region can bind to –> scaffold protein –> can bind to adaptor protein –> can bind to receptor on vesicle or organelle

Question 10

Dynactin

Answer 10

Dynactin is an adaptor protein for Dynein

1. Microtubule –> bound to stalk of dyneine –> AAA motor head –> Dynein tail (which usually interacts with cargo) –> bound to dynactin –> bound to spectrin and ankyrin actin-associated proteins –> bound to transmembrane receptor in vesicle or organelle.

Question 11

Transport of vesicles in Neurons

Answer 11

Involves Kinesin, Dynein, and Mysoin V

1. Vesicles travel from cell body to cell periphery via microtubules
2. Once they reach cell periphery or growth cone, they transfer to actin filaments which are abundant in this location
3. To make a return trip, they must transfer from actin to MT again

Question 12

Transport of pigment granules

Answer 12

1. Background
   
   1. Melanin pigment is in vesicles called melanosomes
   2. Redistribution of pigment vesicles aka melanosomes throughout cell requires transport along MTs and actin
   3. In humans, melanosomes are transported to cell periphery via actin (myosin V) and MTs where they are taken up by keratinocytes
   4. Mutation in transport prevent melanosomes in keratinocytes and results in silvery hair and pigmentation problems in the skin
2. Transport process
   
   1. Myosin Va head binds actin
   2. Myosin Va tail binds to Melanophilin (Mlph)
   3. Melanophilin binds RAB27a GTP
   4. Rab 27aGTP binds to receptor on Melanosome
   5. ***Mutation in Myosin V, Mlph, Rab result in pigmentation problems

Question 13

Myosin Vb

Answer 13

1. Involved in transporting vesicles carrying receptors into dendritic spines
2. Associated with long-term potentiation
   
   1. With more receptors brought to dendritic spines, synaptic changes result in learning and memory.