Molecular Motors II

Question 1

Explain the Cytoplasmic Dynein ?

Answer 1

1. Trafficking of multiple cargo (Membraneous compartments, mRNA, specific proteins, viruses, aggregated proteins and nuclei
2. Moves towards minus ends of microtubules
3. Binds to α-β tubulin dimer
4. Large multi-subunit protein complex composed of two identical heavy chains and multiple light chains
5. N-terminal tails
   - dimerisation/LC binding/cargo binding
6. Linker
   - lever arm
7. C-terminal AAA+ ring and coiled coil stalk
   - Motor and microtubule binding

Question 2

Explain the distinct motor of cytoplasmic dynein ?

Answer 2

• Motor different to kinesin/myosin
• Six repeated AAA+ domains
• Folds to form a linked hexamer
• Coiled-coil domain with microtubule-binding domain (MTBD) pokes out from AAA+ ring
  Linker domain runs across face of AAA+ hexamer
• Tail dimerises two dyneins (flagella forms are monomeric)

Question 3

Explain the binding in a Cytoplasmic Dyenin ?

Answer 3

• ATP binding by AAA+ ring causes conformation change
• Relative position of linker and coiled-coiled change
• Communication to MTBD (microtubule binding domain)
• Binding of ATP weakens binding to microtubule
• Number of ATPs required unclear

Question 4

Cytoplasmic Dynein –motors move in 16 nm steps. It’s active as ?

Answer 4

• Active as a dimer
• Processive Motor with high duty ratio
• Labelling of one motor of a dimer reveals hand-over-hand mechanism
• However, step size more variable and back steps frequent

Question 5

On average, centre of dimer moves in 8 nm steps whilst motor moves ?

Answer 5

In 16nm steps

Question 6

What is the Axial path for kinesin ?

Answer 6

Axial path for kinesin is straight –sticks to one protofilament

Question 7

What is the Axial path for dynein ?

Answer 7

Axial path for dynein is more variable – can step between protofilaments

Question 8

Explain the Flagella ?

Answer 8

• Used for propulsion of both bacterial and eukaryotic cells. Rotate.
• Flagellate bacteria . Eg. Salmonella
• Flagellate eukaryotic cell. Eg. Sperm cell
• Cilia –similar to flagella in composition (generally shorter). Beat back and forth

Question 9

Microtubules in Cilia & Flagella in Eukaryotes ?

Answer 9

• MTs are the fundamental structural unit in cilia and flagella
• Form the Axoneme

Question 10

The dynein “cargo”in cilia movement is ?

Answer 10

The A-tubule, moves along the B-tubule

Question 11

Molecule motors in intracellular transport –Core similarities ?

Answer 11

• Motion requires the hydrolysis of ATP
• Motors evolved to move on either actin or microtubules
• Directional
• Action of two motors
• Hand-over-hand motion
• Processive

Question 12

What class is muscle myosin?

Answer 12

Muscle myosin is of the myosin II class

Question 13

Explain Myosin V in depth ?

Answer 13

• Membrane trafficking
• Moves towards plus ends of actin
• Most studied myosin
• Dimer of two heavy chains (200kDa) and multiple light chains
• N-terminal globular myosin motor domain ATPase/motor
• Six IQ motifs with binding sites for light chains - three times longer than in myosin II
• Rigid lever arm/regulation
• Coiled coil domain
• C-terminal globular cargo binding domain and further light chains
• Active as a dimer (of heavy chains)
• Processive Motor with high duty ratio

Question 14

What does the ATPase cycle brings about ?

Answer 14

Conformational change in converter domain

Question 15

What does the light chain-binding region serve as in myosin ?

Answer 15

It serves as a lever arm to amplify movements of the converter domain, which transitions between pre-stroke and post-stroke configurations

Question 16

For myosin II, short lever arm results in ?

Answer 16

10nm displacement of lever arm

Question 17

For myosin V, longer lever arms result in ?

Answer 17

~37 nm displacement of lever arm

Question 18

Explain Myosin VI in depth ?

Answer 18

1. Membrane trafficking
2. Moves towards minus ends of actin
3. Dimer of two heavy chains and multiple light chains
4. N-terminal globular myosin motor domain plus special insert domain
   - ATPase/motor
5. Two IQ motifs with binding sites for light chains
   - Rigid lever arm/regulation
6. Proximal tail
   - lever arm extension
7. Medial tail coiled-coil
8. C-terminal distal tail/cargo binding domains

Question 19

What does the Inserted region cause ?

Answer 19

Light chain redirects the myosin VI light chain-binding region in a direction opposite to that of myosin V

Question 20

Explain the motion of molecular motors ?

Answer 20

• Motion is coupled to NTPaseactivity
• Motion is produced by conformational changes
• Motion is “actioned” by changes in affinity for binding partner eg. for Microtubules
• Changes in affinity are related to the conformational changes
• Conformational changes are driven by NTP hydrolysis and release of products.

Question 21

What are some diseases associated with deficient motor proteins ?

Answer 21

1. Charcot Marie Tooth disease type 2A (human) :
   - Mutation in Kinesin KIF1Bβ which is specialised to transport mitochondria, leads to a progressive loss of muscle tissue
2. Motor neuron disease (Amylotrophiclateral sclerosis ALS):
   - Mutations in cytoplasmic dynein in MICE leads to ALS (e.g. Legs at odd angles)
3. Griscelli disease (human):
   - Mutations in the human myosin V gene result in ataxia, light pigmentation and a variety of immuno-deficiencies and neurological based symptoms
   - Impaired transport of melanosomes
   - Impaired neuronal function and immune system
4. Spinal muscular atrophy with lower extremity predominance (SMALED)
   - 30 missense mutations in dynein identified in human patients.