Lecture 27 - Actin polymerisation in cell migration

Question 1

What is herceptin and what is its significance?

Answer 1

It is a humanised monoclonal antibody targetting the EC domain of ErbB2, and is used as an anti-tumour therapy for breast cancer.
Acts by binding to inactive ErbB2, preventing its dimerisation by masking the neccessary motif. It also forms heterodimers with other receptor like proteins (competitive inhibition).

Question 2

Is the assembly of actin reversible?

Answer 2

Yes

Question 3

What is the problem with single filaments of actin as structural support and how is this overcome?

Answer 3

Single fibres (YES, FIBRES YOU YANKEE) are structurally weak and often associated with contractile units with myosin.
It is overcome by having shorter but cross-linked actin fibres.

Question 4

Name some properties of actin

Answer 4

Actin is an ATPase & is polarised
-a negative end (pointed)
-a positive end (barbed)
Different types of actin are present

Question 5

At which end is the polymerisation of actin faster/slower?

Answer 5

Polymerisation is faster at the positive end relative to the negative end.
Actin affinity for one another decreases with ATP hydrolysis and dissociation occurs.
Depolarisation is faster at the negative end relative to the positive end.

Question 6

What are the three phases of actin polymerisation, and what are they catalysed and regulated by?

Answer 6

-Nucleation: actin dimer formation, to trimer, to polymer etc. A time consuming process.
-Elongation: Rapid net increase in length, especially at the positive end.
-Equilibrium: no net increase, both poly- and depolymerisation occur.
Regulated by - profilin and cofilin
Catalysed by - Arp2/Arp3 complex & formin.

Question 7

What are lamellopodia?

Answer 7

It is a thin sheet of cytoplasm filled with a criss-cross network of actin filaments, called a dendritic network. Its formation pushes the cell membrane forward, and is dissociating from behind as it elongates.

Question 8

What does a moving cell look like?

Answer 8

A moving cell is (chinese) fan shaped, with tiny finger like projections at the outer edge (filapodia). The nucleus is at the handle, and the lamellopodia is found towards the outer edge as a band. Stress fibres trail behind lamellopodia and provide contractile support. Stress fibres are connected by myosin.

Question 9

What are filapodia?

Answer 9

Rod-like protrusions filled with linear actin filaments. Always associated with lamellopodia.

Question 10

What are stress fibres?

Answer 10

Stress fibres trail behind lamellopodia and provide contractile support. Stress fibres are connected by myosin.

Question 11

Describe the role of formin.

Answer 11

Formin acts as the nucleation point for actin. It induces the direction of polymerisation.

Question 12

Describe the role of the Arp2/Arp3 complex

Answer 12

The Arp2/Arp3 complex requires a nucleated filament to act. Induces the formation of a dendritic network, crucial for lamellopodia formation. Cannot form a nucleation point.

Question 13

What is formin regulated by?

Answer 13

It is regulated by a GTPase called Rho, which is active with GTP and inactive with GDP.
Like Ras, it has high affinity for both, and doesn’t dissociate easily.
While active, it will bind to formin.
It needs a GEF to exchange a GDP with GTP.

Question 14

What is the limiting phase of polymerisation?

Answer 14

Nucleation phase

Question 15

How is polymerisation stopped?

Answer 15

After a certain length, polymerisation is stopped via a capping protein which prevents further elongation.
Formin prevents the capping protein from binding to actin.

Question 16

Can nucleation occur with Arp2/Arp3 complex alone?

Answer 16

No, it promotes nucleation, but formin is still needed.

Question 17

Name 3 nucleation promoting factors

Answer 17

WASP
WAVE
SCAR
They bind to the positive end of actin to induce nucleation for branch formation, allowing dentritic network formation.

Question 18

What inhibits the Arp2/Arp3 complex?

Answer 18

GMF

Question 19

What are the factors of elongation regulation?

Answer 19

Thymosin
Profilin
Cofilin
They act by binding to actin monomers

Question 20

Describe the action of thymosin

Answer 20

Forms a thymosin-actin complex with free actin monomers. The complex cannot bind to actin filaments. It binds actin in the form of β-thymosin.
It prevents polymerisation.

Question 21

Describe the action of profilin

Answer 21

Forms a profilin-actin complex, rapidly adds actin to the positive end of actin filaments.
It cannot bind to the negative end.

Question 22

What is the role of ADF?

Answer 22

ADF is actin depolymerisation factor, and forms an ADF-profilin complex.
It then binds to the actin filament and causes actin dissociation.

Question 23

What is the critical concentration in regards to actin formation?

Answer 23

The rate of polymerisation depends on the free actin monomer concentration. The critical concentration is the concentration of free actin monomers at which association and dissociation is equal (equilibrium phase)

Question 24

How can the rate of polymerisation be regulated?

Answer 24

By controlling the free actin monomer concentration.
They cannot bind to actin filaments if they are associated with another unit.
(β-thymosin for example)

Question 25

Describe the role of profilin not involved in polymerisation

Answer 25

It can also bind to PIs at the cell membrane, promoting the exchange of an ADP with an ATP, acting like a GEF (AEF in this case).

Question 26

What do thymosin and profilin both compete agaisnt each other for?

Answer 26

Free actin monomers.

Question 27

What is the role of an ADF-cofilin complex in dentritic network formation?

Answer 27

If it binds to an actin filament, it breaks in half at that point. Does so to increase the density of actin, enhancing branch formation and forming the criss-cross binding.

Question 28

What are the three types of actin filaments?

Answer 28

Contractile bundles
-actin filaments linked by α-actinin. Space in between allows for myosin attachment for contractility. Found on the very outer edge of the dendritic network (contractile bundle) and as stress fibres (stress bundles).

Parallel bundles
Called fimbrin filaments - like contractile bundles, but no space in between and no myosin. Found in filopodia.

Gel-like network
Cross-linked actin filaments in the dendritic network with filamin at the intersections. Makes up the dendritic network.

Question 29

Describe capping proteins

Answer 29

1 capping protein is needed per 7 actin monomer subunits.
Can be removed upon binding to PIs.
Capping stabilises the filament, preventing depolymerisation.