Microtubules

Question 1

What are Microtubules

Answer 1

• Long hollow cylinders made of the protein tubulin
• much thicker shaper than actin
• made up subunits called: alpha and beta tubulin to make a heterodimer -> they come in together not a single subunits
• alpha tubulin is always GTP bound (like actin with atp)
• beta tubulin cycles between GTP and GDP bound forms
• microtubules are formed from 13 protofilaments that together to make a tub-shaped polymer
• microtubules are also polar (due to different subunits, beta at minus end)

Question 2

Seam

Answer 2

Lateral contacts are alpha-alpha/beta-beta, except at the seam
- seam has been hypothesized to be a point of microtubules formation or disassembly

Question 3

How the GTP-GDP cycle of beta tubulin regulates polymer stability

Answer 3

• beta tubulin will be preferentially added in the GTP bound form at the plus end
• there is a random loss of GTP cap
• catastrophe occurs, where there is really fast shrinkage of the microtubules at the plus end
• rescue occurs, regain of GTP cap, rapid growth occurs

Question 4

Dynamic instability

Answer 4

Property of growth and shrinkage at plus end is called dynamic instability
Filaments with a GTP cap: stable
Without cap: not stable

Important when microtubules are in mitosis

Question 5

Visualization of microtubule dynamics in live cells

Answer 5

• tagging tubulin with GFP, growing and shrinking observed

Question 6

Nocodazole

Answer 6

Interferes with microtubules polymerization, binds beta tubulin

Question 7

Taxol

Answer 7

Stabilizes microtubules, preventing their dynamics, binds beta tubulin

Question 8

Microtubules are nucleated by gamma-tubulin-containing complexes

Answer 8

• is associated with other accessory proteins that form that gamma TuSC, they form a ring that is going to provide a template to build a new microtubule
• gamma tubulin ring complex is anchored microtubule organizing center (MTOC)
• only at the minus end
• remains bound to the minus end to prevent disassembly/growth of microtubule

Question 9

Primary MTOC is eukaryotic cells is the centrosome

Answer 9

• is composed os 2 centrioles and pericentriolar material
• gamma turcs are embedded in the pericentriolar
• minus ends of MTs remain attached

Question 10

Centrioles

Answer 10

• are composed of short microtubules triplets
• sas-6 protein is important in the organization of the centriole
• you can label centrioles, using an antibody called antipericentrin
• not all microtubules anchored to centrosomes in animal cells
• fungi and plants don’t have centrioles

Question 11

Kinesin-13

Answer 11

• induces catastrophe -> depolymerization
• uses its motor ability to pry apart protofilaments even where there is a GTP cap

Question 12

XMAP215

Answer 12

Polymerization factor; binds to free dimers and escorts them the plus end

Question 13

Katanin

Answer 13

• dimeric enzyme that will sever microtubule using ATP

Question 14

Two motors that work with microtubules

Answer 14

Kinesin 1 (conventional Kinesin)
- also has motor domain at N-terminus and coil-coil region
- most move towards the plus end (except 13 which uses ATP to hydrolysis to depolymerize microtubules and Kinesin 14 which is minus end directed)
- tails can either attach to other Mts or cargo (vesicles)

Question 15

Kinesin is ATP cycle driven

Answer 15

• lagging motor domain is hydrolyzed from ATP to ADP, and will change conformation and move over the leading head
• ADP on leading leaves, and ATP comes in

Question 16

Kinesin is ATP cycle driven

Answer 16

• lagging motor domain is hydrolyzed from ATP to ADP, and will change conformation and move over the leading head
• ADP on leading leaves, and ATP comes in

Question 17

Dyneins

Answer 17

• minus end directed
• have heavy, intermediate, and light chains
• also have ATPase activity

Question 18

ATP cycle drives dynein motor movement

Answer 18

• post power stroke, relaxed dynein
• microtubule binding domain released
• stalk swings forward and rebinds along microtubule
• power stroke: motor moves towards minus end of microtubule
• atp hydrolysis causes microtubule attachment
• release of ADP + Pi causes a large conformational change that leads to a power stroke

Question 19

Gliding assays

Answer 19

• you can label just one end
• can used this assay to test directionality of motors or motor speed

Question 20

Cell types use dynein and Kinesin for directional traffic

Answer 20

Can be arrange in different ways to transport things

Question 21

Cell types use dynein and Kinesin for directional traffic

Answer 21

Can be arrange in different ways to transport things

Question 22

Cilia/flagella: specialized microtubule-based structures

Answer 22

Cilia/flagella: specialized eukaryotic cell structures/organelles that use microtubules and dynein
- can be motile or non motile
Motile cilia important for cell movement or movement of fluids (sperm)
Cilia used ciliary dyneins