Actin

Question 1

What are the roles of actin?

Answer 1

cell motility
contractility
shape changes
cytokinesis
cell polarity
phagocytosis
macropinocytosis

Question 2

Overall what is Actins distrubution in the cell?

Answer 2

20% in skeletal

5% of all the proteins in non muscle

Question 3

In non-muscle cells, where are the different actin isoforms, alpha, beta, and gamma found?

Answer 3

gamma- at he periphery

alpha- in STRESS fibers

beta- in ruffles, where cell distorts to move. More UPREGULATION of beta actin means more movement

Question 4

What are some properties of actin?

Answer 4

375 amino acids

1) 43 kDa
2) globular monomer, with two domains, G-actin

3) At least 6 isoforms in mammals, encoded by separate genes

Question 5

What are the three classes of actin?

Answer 5

Class 1 - Non-muscle beta, gamma and smooth muscle gamma-actin.

Class 2 - Skeletal, cardiac and vascular a-actin

Class 3 - Actin-RPV, centractin, lower eukaryote actins

Question 6

Name all of the actin structures in the cell (labile, stable, and the junctions.)

Answer 6

Transient/labile surface:
Lamellipodia
Filopodia
Ruffles

Stable actin structures:
Microvilli (contain bundles of 20 actin filaments
stabilised by cross-linking proteins (the bundling proteins
villin, fimbrin and espin))

Actin associated junctions:

• focal complexes
• focal adhesions

Question 7

How is actin assembled?

Answer 7

Process requires energy 
from nucleotide (ATP-ADP) hydrolysis in cells. 

First spontaneously assembles from G-actin subunits around a nucleus (forming F-actin). When it is polmerized it binds to ATP (in its binding site). It is elongated. When it depolymerised ATP is hydrolysed to ADP.

Actin binding proteins regulate monomer to filament ratio
by exploiting the difference in the assembly characteristics at either end (the polarised positive and negative ends)
Here different kinetics are shown; slow growing-end is at the minus-end and the fast growing-end is at the barbed plus-end. As a result it grows in a arrowhead pattern.

Cell signalling alters actin assembly such as small GTPases.

Question 8

What is the structure of actin?

Answer 8

Trimers and elongates by addition of monomers which forms either filaments or networks/bundles.

6-8nm wide

Question 9

When do actin filaments form?

Answer 9

As a result of:

1) Uncapping
2) De novo nucleation
3) severing existing filaments

Question 10

Describe actin formation tread-milling.

Answer 10

At the positive end less actin subunits needed to grow the filament, but a higher subunit concentration is needed if the negative end (pointed) is to grow and so growth mainly at positive barbed end.

Subunits are added at positive end and LOST AT NEGATIVE END and the two ends grow UNEQUALLY (tread-milling)

Question 11

Describe the three stages of actin filament growth

Answer 11

1) Lag phase
2) polymerisation phase
3) steady state with treadmilling

Question 12

How is free G-actin (used as subunits) maintained?

Answer 12

Regulatory proteins control assembly and keep
G-actin concentration high (50% of all actin in this state)

eg, 1)Profilin

2) Capping protein- caps the positive barbed-end of he F-ACTIN , stops elongation as free G-actins cannot bind

Tropomodulin binds to minus end.

3) Thymosin-b4- binds to G-actin making it polymerisation-incapable as there isnt a high enough concentration of free nucleotides

Question 13

What is Profilin?

Answer 13

Regulatory protein of actin that binds to the G-ACTIN, stops it being added, limits elongation.

It is a Ubiquitous protein

It also associates with the barbed end (+) of actin filaments. This effectively reduces the critical concentration.

Too much and too little profilin is detrimental to filament formation.

Knockouts in mice and yeast show it to be an essential protein.

Question 14

What is beta-thymosin

Answer 14

Regulatory protein of actin, found at the cell periphery that binds to the ATP-ACTIN (the free G-actin) moreso than the ADP-ACTIN, so when positive end is free actin is ready to assemble.

Question 15

What are the two concentrations needed at either end of the actin filament

Answer 15

0.1 µM @ positive versus 0.6µM @negative

Question 16

Give some examples of ACTIN binding proteins.

Answer 16

50 different types
a-actinin used for bundling

tropomyosin used for side binding

Cofilin used for severing filament

Question 17

What is a formin?

Answer 17

Protein that helps actin assembly through the reloading of its whiskers with actin subunits

Question 18

What role does the binding protein filamin have?

Answer 18

Filamin forms a cross-linked actin network at the cell cortex

Question 19

What is a ERM protein and what does it do?

Answer 19

Ezrin Radixin Moesin: ERM proteins link the actin filaments to the plasma membrane by binding to the filament and a membrane protein.

Question 20

There are many actin crosslinking proteins

Answer 20

1) fimbrin
2) a- actinin
2) spectrin
4) dystrophin

used for different fibers and functions; depending on their size and shape, they have differing interactions with the actin

Question 21

What mediates the signalling that regulates actin state?

Answer 21

Ras superfamily of proteins

1) GTPase Rac (in meshwork lamellipodium)
2) Rho (in contractile stress fibers)
3) cdc42 (in parallel filopodium)

stimulated by growth factors, ECM and other hormones

Question 22

Describe two models of proposed Actin assembly

Answer 22

1)Treadmilling model :
depolymerisation only at the pointed ends, occurs throughout
the entire filament array and is necessary to allow
treadmilling
growth of filaments from existing actin filament population
barbed ends concentrated at leading edge, pointed ends
throughout the cytoplasm (depolarising at the rear)

2)Dendritic brush model :
new actin filaments nucleated ON existing filaments at the leading edge,
capping of pointed ends of actin filaments
requires actin depolymerisation, releasing filaments.

Question 23

What is the Arp2/3 complex?

Answer 23

consists of 7 polypeptides (Arp2 and Arp3, p40, p35, p21, p18 and p14(ARPC1-5) sharing similar structure/genes/homology to actin

It binds to and caps the pointed-end (negative) and can act as a start point for assembly (its nucleation point)

This enables assembly at barbed-end, binds to side of pre-existing filaments to produce BRANCHING (forms branched webs in lamellipodia)

It is in high concentration at leading edge.

ARP2/3 nucleates new filaments, caps pointed-ends and anchors them to pre-existing filaments (at y junctions)

It can be activated by signalling and regulatory proteins (NPFs-nucleation, Rho-family GTPases (cdc42, Rac, Rho) and actin polymerisation promoting factors like WASP,WAVE)

Rho-GTP stimulates actin growth and stress fibers

Question 24

What effect can pathogens have on actin?

Answer 24

Listeria, Shigella, Helicobacter and several other pathogens hi-jack the actin machinery to promote infection such as bacterial escape from phagocytosis.

Question 25

What is the role of the Rho-family GTPases (cdc42, Rac, Rho) in cell movement?

Answer 25

If RHO is activated, myosin 2 is activated at the back of the moving cell

If RAC is activated, APR is activated at he front of the moving cell

cdc42 is activated at the front of the moving cell.

Treadmilling of the actin filaments in the leading edge will help cell migrate.

Question 26

In summary what are the factors that control actin and its protrusion of the cell?

Answer 26

1) cell surface receptors
2) Rho-family GTPases, Cdc42, Rac
3) WASP and Scar related proteins
4) Arp2/3 complex
5) capping protein
6) profilin
7) cofilin (ADF)