Ostap - Myosin

Question 1

How many myosin genes in humans?

Answer 1

38 genes, 12 families

Question 2

Domains of myosin

Answer 2

1. Tail
2. Regulatory domain - small light chains attach to this (similar to calmodulin)
3. Motor domain- provides chemical energy to do work, has nucleotide binding site. Linking/scaffolding domains exist here

Question 3

In vitro motility assay**

Answer 3

First developed for the myosin family. Take a glass coverslip, coat it with nitrocellulose and myosin, block it. Add actin. Then add ATP. You can drive motility of actin filaments.by doing this.

Question 4

What can you treat with to cut up myosin?

Answer 4

Chymotrypsin - will cut >– ——

Papain: will cut the 2 heads apart.

Question 5

What domains do you need to bind (myosin 2)

Answer 5

Motor domain and regulatory domain

Question 6

Myosin 2

Answer 6

Present in muscle

Question 7

Do we have a high res image of actin bound to myosin?

Answer 7

No.

Question 8

Rigor complex

Answer 8

Myosin catalytic domains bind tightly to actin in the absence of ATP. (This is responsible for rigor mortis). THis interaction is weakened by ATP, counterintuitively.

Question 9

Which direction does myosin move toward?

Answer 9

The barbed end of actin filaments

Question 10

Velocity

Answer 10

Velocity = stroke distance/time

The neck region of the myosin motor domain acts as a lever arm to generate movement.

Question 11

What is the relationship between velocity and lever arm length?

Answer 11

Linear - as lever arm length increases, velocity increases.

Question 12

Relationship between myosin’s ATPase activity and actin?

Answer 12

As you add more actin you increase the ATPase rate until you reach a plateau.

Question 13

Steps in force generation

Answer 13

Detach –> Reverse power stroke (weakly bound states) –> Re-attach –> Power stroke

Question 14

What is duty ratio? What duty ratio does myosin have?

Answer 14

Duty ratio = time in force bearing state/total ATP-ase time

Myosin with high duty ratio- spends lots of time in strongly bound state
Myosin with low duty ratio spends lots of time detached from actin

Question 15

Functions of the myosin II family

Answer 15

Skeletal muscle
Cardiac muscle
Smooth muscle
We mostly have myosin 2

Question 16

Myosin

Answer 16

Tick filament

Question 17

Actin

Answer 17

Thin filament

Question 18

Titin

Answer 18

Connects myosin to the Z disk

Question 19

Z line

Answer 19

Attaches to actin

Question 20

Myosin II

Answer 20

Composed of 2 heavy chains and 4 light chains. 6 chains total.

Question 21

Is myosin 2 unipolar or bipolar?

Answer 21

Bipolar

Question 22

Where are barbed ends of actin in muscle?

Answer 22

Anchored at Z disk.

Question 23

HOw does regulation work? What about contraction?

Answer 23

RElaxation - Regulatory proteins inhibit actin-myosin interactions. Few heads are bound to actin. Sarcomere can be stretched passively.
Contraction - muscle activated by actin. Thousands of sarcomeres shorten in series causing muscle to shorten. ATP is hydrolyzed anf foce is produced.

Question 24

What experiment could you do to study muscle contraction and calcium?

Answer 24

Remove surface membranes from a single muscle fiber, measure isometric tension in presence of ATP and calcium concentrations.

Question 25

Tropomyosin

Answer 25

40 nm long coiled coil of 2 beta helical polypeptides

Question 26

Troponin

Answer 26

3 proteins:
TN-C - binds calcium and is related to calmodulin
TN-I - inhibits actin myosin interaction
TN-T - binds tropomyosin.
(THese are the 3 subunits of troponin).
In the absence of calcium tropomyosin binds myosin binding site.

Question 27

How does calcium mediate muscle contraction

Answer 27

Basically tropomyosin blocks the myosin binding site, and then myosin binding site is exposed by Ca mediated tropomyosin movement.

Question 28

Are muscle myosin II motors high or low duty?

Answer 28

LOW DUTY.
Weakly bound - little time in strongly bound states.
however…Smooth muscle - these hold contraction a long time. ADP release is slower for smooth muscle myosin.

Question 29

What’s an optical trap?

Answer 29

Laser/tractor beam. Infrared laser beam that you can use to trap a bead or anything else that’s refractive. You trap a particle and move it around. Laser acts as a spring and you can calculate the spring constant. You can use this to measure single molecule interactions.
measures piconewton forces, nm displacements, rapid kinetics.
Sustained force from smooth muscle. Attachment is really long.

Question 30

Blebbistatin

Answer 30

Myosin II inhibitor

Question 31

How is myosin II related to glioma?

Answer 31

Affects glioma invasion in the brain and migration through the matrix. Also it is a tumor suppressor in squamous cell carcinoma. Removal gives you more.

Question 32

What about non muscle myosin II in cells?

Answer 32

Forms bipolar filaments, regulated by light chain phosphorylation.No troponin but there is tropomyosin.

Question 33

Myosin V

Answer 33

WE have 3 of these. 2 motor domains - 2 headed. 12 light chains bound to regulatory chain. Light chains associated with tail domain. Coiled coil responsible for dimerization.

Functions
Vesicle and organelle transport, mrna delivery

High duty ratio.

Must stay associated to cytoskeletal track during transport
Must regulate motor activity/cargo attachment
Must bind tightly and specifically bind to cargo

Rate limiting step is ADP release - both heads have high duty ratios.

Processive -takes multiple steps along actin filament without dissociating.

36 nm steps.

Question 34

How long is the actin helix

Answer 34

36 nm, same size as myosin v step

Question 35

Myosin VI

Answer 35

High duty ratio
Pointed end directed motor activity.
Processive
2 calmodulins bind but only 1 IQ domain
THe lever arm allows end directed motility.
Dimerizes when it’s croweded together - cargo mediated dimerization.
Takes frequent backward steps.

Edocytosis, autophagy