Lecture 19 & 20 - Actin

Question 1

Actin filaments (also known as ________) are ______ ______ of the protein actin

Answer 1

microfilament; helical polymers

Question 2

Actin subunits

Answer 2

also called globular or G-actin
- polypeptides carrying a molecule of ATP or ADP

Question 3

Is actin and ATPase?

Answer 3

yes; actin subunits have ATP, actin filaments hydrolyze this ATP into ADP

Question 4

What provides polarity to the filament?

Answer 4

the way the actin subunits assemble
- at minus end (pointed end), the ATP-binding pocket is exposed
- at plus end (barbed end), the ATP-binding pocket is buried

Question 5

Where does most growth happen on a filament?

Answer 5

from the (+) end

Question 6

What is the rate-limiting step in the formation of actin filaments?

Answer 6

nucleation => very slow
- doesn’t happen in a cell

Question 7

How many actin molecules are needed to make the growing complex more stable?

Answer 7

3 actin monomers; 2 actin molecules bind relatively weakly to each other

Question 8

What gives the appearance of the filaments moving in the (+) end direction?

Answer 8

actin monomers are joining the (+) end while other are falling off the (-) end

Question 9

What process pushes membranes forward?

Answer 9

actin treadmilling

Question 10

What drug stabilizes F-actin?

Answer 10

phalloidin => cannot grow or shrink actin, we die

Question 11

What are some examples of structures assembled from actin filaments?

Answer 11

• microvilli
• cell cortex
• adherens belt
• filopodia
• lamellipodium/ leading edge
• stress fibers
• contractile ring

Question 12

What are the 2 structures that migrating cells make?

Answer 12

protrusive structures termed filopodia and lemellipodia

Question 13

What do actin-binding proteins do?

Answer 13

they regulate polymerization and length of filaments
- monomer-sequestering proteins bind to actin monomer and prevent it binding to filament
- actin-polymerizing proteins
- filament-severing proteins cut filaments
- filament-capping proteins prevent dissociation

Question 14

CapZ

Answer 14

a capping protein; prevent G-actin addition and loss
- binds to (+) end
- looks almost identical to G-actin but lacks actin binding site for other G-actin subunits

Question 15

Cofilin

Answer 15

cuts actin into small pieces

Question 16

What does actin filament crosslinking do?

Answer 16

makes complex structures

Question 17

Alpha-actinin

Answer 17

a crosslinking protein that makes F-actin bundles
- a dimer
- crosslinkers must have 2+ actin binding sites
- rigid

Question 18

Filamin

Answer 18

a flexible actin crosslinker protein
- crosslinkers must have 2+ actin binding sites
- a dimer
- flexibility allows different angles

Question 19

Arp2/3 complex

Answer 19

promotes actin branching
- composed of 7 subunits structurally similar to G-actin
- initiates a new branched filament by binding to the side of the filament and recruiting actin monomers

Question 20

What links the actin cytoskeleton the membrane?

Answer 20

ERM proteins
E= Erzin
R = Radixin
M = Moesin
- binding sites for actin and membrane proteins are hidden but revealed when Erzin is phosphorylated

Question 21

How do cells assemble complex actin structures?

Answer 21

by dynamically changing the activity and localization of actin-binding proteins

Question 22

How do actin binding proteins become activated?

Answer 22

with a signal
- Rho GTPases are the signal (Cdc42, Rho, Rac)
- on state is GTP bound
- off state is GDP bound

Question 23

What do Rho GTPases do?

Answer 23

they coordinate the activation of all actin-binding proteins required to build a specific cytoskeletal arrangement
- each Rho GTPase protein signals the formation of a different actin structure
- they locally activate actin-binding proteins

Question 24

Profilin and Formin

Answer 24

profilin: a small protein, key regulator of actin polymerization
formins: a group of proteins involved in the polymerization of actin and associate with the fast-growing end of actin filaments
- grabs monomers and puts them near (+) end

Question 25

Where are filopodia and lamellipodia present?

Answer 25

at the leading edge of migrating cells

Question 26

What pushes the cell forward?

Answer 26

actin polymerization and branching
- researchers removed the nucleus to see if transcription was involved, cell still moved

Question 27

What are the motor proteins that walk on actin filaments?

Answer 27

myosins; transport a wide variety of cargo; moves towards (+) end

Question 28

Myosin power stroke

Answer 28

1. Without ATP myosin is attached to actin filament in rigor => myosin head is stuck to actin
2. ATP binding to myosin head domain causes the release of actin filament
3. ATP is hydrolyzed causing a large conformational shift in the ‘lever arm’
4. Release of phosphate & myosin head binds to actin
5. power stroke is the force generating step
6. ADP is released, myosin head remains tightly bound (rigor state)

Question 29

What part of myosin makes actin move?

Answer 29

the motor domain; without it, the filament does not move

Question 30

What are the 2 types of myosin?

Answer 30

conventional and unconventional
- conventional form bipolar filaments
- unconventional do not form bipolar filaments

Question 31

Myosin V

Answer 31

tethers and transports organelles
- globular tail binds cargo
- dimerization
- neck = Ca2+ regulatory region
- head = actin and ATP binding

Question 32

Myosin 1

Answer 32

has only one head, binds membranes, and powers membrane deformation
- doesn’t move along actin filaments
- does power stroke, but doesn’t really release actin