Molecular Motors I

Question 1

What are some Cytoskeletal Transport Systems ?

Answer 1

• Motor
• Cargo
• Fuel
• Track

Question 2

Explain Molecular Motors ?

Answer 2

Proteins that produce motion

• Utilise(chemical) energy and convert it
• Often utilise conformational change
• Often require chemical energy (eg. ATP, NADP etc)
• Very complex molecular structures

Question 3

What are Microtubules ?

Answer 3

Microtubules are polymeric fibrous proteins and are the tracks along which cargo can be moved

Question 4

Now explain Microtubules in detail ?

Answer 4

• Microtubules are dynamic
• They are formed by tubulin –alpha and beta heterodimer
• They self-assemble into protofilaments
• And associate laterally to form a cylindrical wall
• The hydrolysis of GTP -> GDP allows assembly and GDP is bound weakly. When GDP diffuses out of the binding site, then the microtubules disassemble
• They have polarity –a positive and negative end.

Question 5

Describe the microtubule motility in squid giant axon ?

Answer 5

• Membrane organelles + mitochondria moved anterograde (towards the synapse) and retrograde (towards the cell body) directions at 0.5-2.0 ums-1
• Multiple organelles were seen to move along the same fiber in opposite directions and to pass each other without apparent collision
• Directed motion along linear tracks
• Transport continued normally after the axoplasm was extruded from the giant axon, providing ATP levels were maintained
• Non-hydrolysable ATP analogues (e.g. AMP-PNP) did not support movement

Question 6

What are Molecular Motors Proteins ?

Answer 6

Enzymes which couple energy from ATP hydrolysis to mechanical movement along linear tracks

Question 7

What are the two motors on the microtubule and explain what direction they move in?

Answer 7

• Kinesin I - which moves towards the positive end

- Dynein - which moves towards the negative end

Question 8

What motor is on the actin filament ?

Answer 8

Myosin V which moves towards the positive end

Question 9

When ATP is added, some organelles with motor proteins attached bind to ?

Answer 9

The microtubules

Question 10

Sometimes you see an organelle switch directions –this must ?

Answer 10

Have both motor proteins attached

Question 11

Explain Convention Kinesin - Kinesin 1 ?

Answer 11

1. Transport of membranes (vesicle, organelles, etc)
2. Two heavy chains (120 kDa) and two light chains (70kDa)
3. N-terminal globular head domains
   - ATPase/motor
4. Neck linker
   - power “stroke”/directionality/processivity
5. Coiled-coiled stalk
   - Control
6. C-terminal globular cargo binding and selection domain

Question 12

The discovery of Kinesis ?

Answer 12

• Vale, Schnapp, Sheetz and Reese (1985) separated squid giant axon into soluble and organelle-rich fractions depleted of microtubule protein
• These fractions were combined in various ways to assay for microtubule motility, for example using the “sliding assay”

Question 13

Single kinesin-1 dimer can produce ?

Answer 13

Force coordinated movement of both heads

Question 14

Explain Kinesin-1 dimer/cargo steps ?

Answer 14

• Exact position of cargo (latex bead) measured with nanometre accuracy using “optical trap”
• Staircase profile characteristic of a processivestepping motor
• 8 nm steps between stable states
• Corresponds to spacing between b-tubulinsin protofilament
• Each 8 nm step depends on hydrolysis of one ATP

Question 15

Explain the two alternative models that could account for processive motion using two heads ?

Answer 15

1. Inchworm model:
   - Each head moves in 8 nm steps
   - First foot moves forward then the second one catches up
   - Two head steps moves kinesin 8 nm
2. Hand-over-hand model:
   - Each head moves in 16 nm steps
   - Trailing and leading heads swap
   - One head step moves kinesin 8 nm

Question 16

Explain FIONA ?

Answer 16

Fluorescence Imaging with One Nanometre Accuracy

• Label motor of one head with fluorophore
• Monitor fluorescence using microscope
• Follow individual fluorophores
• Theoretically, the centre can be determined with 1 nm precision by collecting a sufficient number of photons.

Question 17

Explain in detail the Conventional kinesin - head and neck linker form the motor?

Answer 17

• Kinesin shares a catalytic motor core with the myosins -both are member of the G-protein family of NTPases
• Different polymer binding elements –in kinesin a ~12 amino acid loop binds tubulin whereas in myosin a ~140 amino acid domain binds actin
• Different mechanical elements provide the power stroke
• In myosin, the converter domain and lever arm
• In kinesin, the shorter neck linker
• Different dependence of polymer binding on nucleotides

Question 18

Sensor, comprising Switch I and Switch II, senses ?

Answer 18

The presence of absence of the terminal (γ) phosphate group

Question 19

Switch II acts as ?

Answer 19

A spring loaded gate, moving in or out depending on the presence of the g-phosphate

Question 20

ATP binding engages the Sensor, phosphate release ?

Answer 20

Disengages the Sensor

Question 21

Small movements of Sensor are transmitted to other parts of the protein via ?

Answer 21

The relay helix

Question 22

Movement of the relay helix changes ?

Answer 22

• The conformational of the neck linker and the microtubule binding domain
• Small nucleotide-driven conformational changes are amplified