Cytoskeleton Actin

Question 1

The cytoskeleton is made up of?

Answer 1

Actin, microtubules, intermediate filaments

Question 2

Functions of the cytoskeleton

Answer 2

1. Provide shape/structure to the cell
2. Provide connections between parts of a cell
3. Enable cell movement (cell division)
4. Provide tracks for movement of other structures

Question 3

Actin

Answer 3

1. Is an ATP binding protein (has an ATPase fold)
2. F-actin is a helical polymer of actin monomers (g-actin)
3. Polymers are polar
   Plus end = barbed
   Minus end = pointed end

Question 4

Actin nucleation

Answer 4

Actin monomers (g actin) will be assembled into dimers, then trimmers, then filaments (f-actin)
Nucleation: refers to the creation of the first trimer (which is when it becomes stable)

Question 5

What is actin polyemrization?

Answer 5

Nucleation: lag phase (rate limiting)
Elongation is fast (sort of looks like logistic growth model): growing actin filament
Steady state: actin filament with subunits coming on and off

Question 6

How can you accelerate polymerization?

Answer 6

Polymerization is faster if preformed seeds (that are already nucleated) are added
-> monomers are added much more quickly at the plus end

Question 7

Without seeds

Answer 7

Nucleation is longer

Question 8

With seeds

Answer 8

No nucleation phase

Question 9

Pyrene actin Assay

Answer 9

Use a version of actin that is going to fluoresce ONLY when it’s present in f-actin
Actin will be fused to fluorescence molecule called pyrene, pyrene will be excited but won’t fluoresce until actin is in conformation of f-actin
Can calculate rates of nucleation and elongation
CAN ONLY STUDY DYNAMICS IN A CONTROLLED ENVIRONMENT

Question 10

Purpose of ATP

Answer 10

• acting has atp fold
• changes kinetics of filament activity
• ATP bound form of actin is going to be preferentially added, this puts actin in a conformational form where addition is very favorable
• ATP will be hydrolyzed to ADP
• rate of association for ADP-actin if higher than ATP

Question 11

Actin treadmiling

Answer 11

• at steady state, ATP-actin added to plus end
• then ATP is hydrolyzed to ADP
• ADP actin is preferentially lost from the minus end
• filament stays roughly the same size due to net addition and subtraction

Question 12

Would more of less actin be present if you
A. Capped the plus end
B. Capped the minus end
C. Binds to monomers and prevents the addition to the + end
D. Severs f-actin filaments in the middle

Answer 12

A. Less
B. More
C. Less
D. Net no change because you’re not changing the relative rates of addition or subtraction

Question 13

Profilin

Answer 13

-binds to monomers and promotes addition to the plus end
- inhibits nucleation
- accelerates alongation
COMPETING WITH THYMOSIN to bind to actin

Question 14

thymosin

Answer 14

• Binds to monomer and prevents addition to the plus end
• it blocks the site of actin that should be binding to the plus end

Question 15

Cofilin (filament severing protein)

Answer 15

• binds along the length of actin
• length is shorter because cofilin causes a change in conformation thats twists the filament, straining it and this causes depolymerization to occur
  (Rapid disassembly of the whole filament)

Question 16

Actin nucleator: Formins

Answer 16

• nucleator linear f-actin filaments
• functions as dimers, move progressively by capturing monomers and adding them to the plus end
• in pyrene actin assay, you can elongation happens quicker
• ENHANCED BY PROFILING

Question 17

Actin Nucleator: ARP2/3 complex

Answer 17

• inactive arp2/3 complex needs activating factor
• back end of complex can bind to the side of an actin filament (branched)
• regulated by WASp
• binds to the minus end

Question 18

RAC-GTP

Answer 18

• activates WASp -> activatesARP2/3 complex
  AND/OR
• activates PAK -> activates filamin

Question 19

Rho-GTP

Answer 19

• activates formins to cause actin bundle growth

Question 20

WASp

Answer 20

• has a lot of protein domains
• VCA at the end
• can auto inhibit (so not active)
• when bound to cdc42 (gtpases), relieve auto inhibition, allows VCA domain to bind to the ARc2/3 complex -> will induce conformational change in each of the 7 subunits
• can also bind to g-actin

Question 21

WASp

Answer 21

• has a lot of protein domains
• VCA at the end
• can auto inhibit (so not active)
• when bound to cdc42 (gtpases), relieve auto inhibition, allows VCA domain to bind to the ARc2/3 complex -> will induce conformational change in each of the 7 subunits
• can also bind to g-actin

Question 22

Actin cross linking proteins

Answer 22

• actin cross linking proteins have more than one actin-binding site
• molecules like fimbrin, alpha-actinin, spectrin, and filamin can organize actin fibers
  NOT COVALENT LINKAGES, regular protein protein interactions