Model for Kinesin Motion

Question 1

What is the general structure of kinesin?

Answer 1

• 2 x 120kDa heavy chains
• 2 x 70kDa light chains
• Coiled coil domain

Question 2

Is kinesin a dimer?

Answer 2

Yes

Question 3

How many regions on kinesin can ATP bind?

Answer 3

2

Question 4

Where does ATPase action occur in kinesin?

Answer 4

N-terminal globular heads

Question 5

Where does cargo bind on kinesin?

Answer 5

On the C-terminal globular cargo binding and selection domain

Question 6

How was a movement assay done for kinesin?

Answer 6

• Isolated purified motor proteins stick on glass via tails with heads sticking upwards
• microtubule attaches to motorhead and is moved towards minus end

Question 7

How was observation of kinesin ‘walking’ first done?

Answer 7

• Microtubule attached to glass cover strip and latex particle moved along
• Dilute motor protein enough so each particle only has 1 motor attached

Question 8

What are the problems associated with the direct observation of kinesin walking?

Answer 8

• Latex particle subject to Brownian motion so never know exactly where particle is on microtubule
• Difficult to see individual steps

Question 9

How can the problems with observing kinesin be solved?

Answer 9

Using an optical trap

Question 10

How do optical traps work?

Answer 10

• Moves at the same rate as the motor
• Focussed beam of laser light traps particle (stops movement due to Brownian motion0

Question 11

What profile does kinesin produce with an optical trap?

Answer 11

• Staircase profile characteristic of a processive motor
• 8nm steps between stable states

Question 12

How does FIONA work?

Answer 12

• Label one motor head with flurophore
• microtubule on cover slip and fluro labelled kinesins added
• Collect enough images of flurophores that the centre of the spot can be found accurately

Question 13

Why was FIONA used in analysis of kinesin?

Answer 13

Can differetiate between 8nm and 16nm steps

Question 14

What model does kinesin use to move along microtubules?

Answer 14

Hand over Hand

Question 15

When does the power stroke occur in kinesin?

Answer 15

When ATP is bound

Question 16

Is kinesin a high duty bound or low duty bound motor?

Answer 16

High duty bound

Question 17

What happens in kinesin when ATP binds?

Answer 17

Neck linker docks to core motor and now faces forwards

Question 18

What happens after the power stroke in kinesin?

Answer 18

ATP hydrolysis

Question 19

What happens when ADP is bound in kinesin?

Answer 19

• Head releases tubulin, neck linker unlocks
• Motor is weakly bound to track

Question 20

What happens when ADP is released in kinesin?

Answer 20

• Binds new tubulin site
• Neck linker facing backwards

Question 21

When happens in kinesin when the neck-linker docking is completed by the leading head?

Answer 21

The partner head is thrown forward by 16nm

Question 22

What does the processive hand-over-hand stepping of kinesin 1 require?

Answer 22

The ATPase cycles of each head are co-ordinated

Question 23

How are the ATPase cycles of the heads in kinesin co-ordinated?

Answer 23

• Core ATPases do not contact each other directly
• Long range allostery
• Mechanical tension transferred between 2 heads via neck linkers