W21

Question 1

Describe contact inhibition of locomotion.

Answer 1

When cells come into contact with one another, they stop migrating and repolarise to move away from each other

Question 2

Describe the filaments responsible for cell migration.

Answer 2

Actin filaments or microfilaments are two-stranded helical polymers of actin

Question 3

Describe actin polymerisation for cell migration.

Answer 3

Monomers preferably bind at the barbed (+) end and are lost at the pointed (-) end. Polymerisation generates force via a Brownian rachet mechanism for membrane protrusion. The actin filament moves constantly via Brownian motion, allowing a new monomer to polymerise, extending the filament to protrude at the plasma membrane

Question 4

Describe the treadmilling-type reaction of protrusive motility driven by nucleotide hydrolysis.

Answer 4

The treadmilling-type reaction is driven by addition of subunits at the barbed end and loss of subunits at the pointed end. ATP-bound actin eventually hydrolyses ATP to ADP. Energy released by hydrolysis destabilises the filament, causing the ADP-bound subunit to preferentially depolymerise

Question 5

Describe the stability of actin polymer formation.

Answer 5

Two actin monomers bind weakly, the addition of a third makes the group more stable (nucleation). Nucleation is the rate-limiting step as a helical polymer is stabilised by multiple contacts between adjacent subunits

Question 6

Describe the action of accessory proteins cofilin and gelsolin.

Answer 6

Sever ADP-bound actin from the filament, making more actin available to polymerise at the barbed end

Question 7

Describe the action of accessory protein thymosin.

Answer 7

Sequesters actin subunits

Question 8

Describe the action of accessory protein profilin.

Answer 8

Catalyses ADP to ATP exchange of actin subunits and presents them to the barbed end for polymerisation to occur

Question 9

What is the function of capping proteins in relation to actin filaments?

Answer 9

Regulate filament length, keeping them short and branched as opposed to long and thin, to improve stability considering actin flexibility

Question 10

What is the collective function of accessory proteins cofilin, gelsolin, thymosin, and profilin?

Answer 10

Regulate the effective concentration of soluble subunits

Question 11

What is the function of the actin cross-linking protein spectrin?

Answer 11

Spectrin (tetramer) binds two cortical actin filaments around the outside of the cell

Question 12

What is the function of the actin cross-linking protein fimbrin?

Answer 12

Organises filaments into parallel tight bundles at filopodia microspikes with the growing ends all facing the same direction

Question 13

What is the function of the actin cross-linking protein alpha-actinin?

Answer 13

Alpha-actinin (dimer) binds two actin filaments anti-parallel, forming loose bundles at the trailing tail of the cell. Myosin II binds between bundles to contract the rear end

Question 14

What is the function of the actin cross-linking protein filamin?

Answer 14

Filamin (dimer) organises actin filaments at 90 degrees to each other, forming a lattice network at the leading edge of the cell

Question 15

Describe filopodia.

Answer 15

Thin actin-rich projections that extend beyond the leading edge, with receptors on their surface that sense the surroundings of the cell

Question 16

Describe lamellipodia.

Answer 16

Form a dendritic array of actin filaments cross-linked and branched beneath the membrane that extend the leading edge in a sheet

Question 17

Describe stress fibres.

Answer 17

Antiparallel bundles of actin filaments that span the length of the cell with alpha-actinin. Stress fibres anchor into adhesion structures at the front of the cell, as cells move over them they become larger and are used as traction to pull up the rear. If blocked, a long trailing tail is formed that becomes detached

Question 18

Describe focal adhesions.

Answer 18

Contain transmembrane integrins, extracellular domain binds the matrix while intracellular domain binds the actin cytoskeleton, involved in traction control of stress fibres

Question 19

Where are lamellipodia found in non-migrating and migrating cells respectively?

Answer 19

Found at the periphery in non-migrating cells and polarised to the leading edge of migrating cells

Question 20

Describe the function of the Arp2/3 complex.

Answer 20

Activated by a nucleation promoter factor, binds the side of a pre-existing actin filament that has been capped via capping proteins. Arp2/3 complex nucleates the formation of a new actin filament at a 70 degree angle, using actin monomers presented to the growing barbed end via profilin, creating a new branch

Question 21

Describe the structure of the Arp2/3 complex and how it interacts with a nucleation promoting factor (NPF).

Answer 21

Comprised of seven proteins, two actin-related proteins (Arp) Arp 2 and Arp3 and stabilised by five other proteins ARPC1-5. NPF VCA domain binds Arp2 and Arp3 alongside an actin monomer, forming a stabilised trimer that nucleates production of a new actin filament

Question 22

Describe the formation of a nascent filopodium.

Answer 22

Some actin filament barbed ends acquire a privileged status by binding a complex of proteins called the tip complex that allows them to elongate continuously without being capped. These barbed ends congregate and elongate together, crosslinked by fascin and fimbrin, to produce a nascent filopodium

Question 23

Describe the function of serine/threonine kinases in actin polymerisation.

Answer 23

PAK is activated by cell-surface receptors and phosphorylates another serine/threonine kinase LIM. LIM phosphorylates ADF/cofilin, inhibiting the severing and depolymerisation of ADP-actin filaments, facilitating a burst of polymerisation without depolymerisation

Question 24

What two domains are conserved in the nucleation promoting factors WASp and WAVE?

Answer 24

C-terminal VCA domain and proline-rich domain

Question 25

Aside from conserved domains, what other domain does the nucleation promoting factor WASp have?

Answer 25

Regulatory CRIB domain, autoinhibits WASp until activated by Cdc42 binding

Question 26

Aside from the conserved domains, what other domain does the nucleation promoting factor WAVE have?

Answer 26

Regulatory SHD domain, binds four proteins Abi1, Nap1, Pir, and HSC300 all involved in the regulation and activation of WAVE. Rac binds Nap1 and Pir, releasing them from the WAVE complex, activating WAVE so its VCA domain is available to bind the Arp2/3 complex

Question 27

Name the Rho family GTPase that is responsible for each actin superstructure.

Answer 27

Rac produces lamellipodia, forming membrane ruffles. Rho produces stress fibres. Cdc42 produces filopodia

Question 28

Describe the effect of actin polymerisation via Rac and Cdc42.

Answer 28

Downstream effectors of Rac include WAVE and PAK, and for Cdc42 includes PAK, WASP, and IQGAP. Rac and Cdc42 are intimately linked so activation of Cdc42 leads to GEFs activating Rac. This means when filopodia form, lamellipodia will follow

Question 29

Describe the downstream effectors of Rho.

Answer 29

ROCK is a serine/threonine kinase that phosphorylates myosin light chain, leading to stress fibre formation. ROCK also phosphorylates MLC-phosphatase, inhibiting it. mDia1 is a formin protein that acts as a leaky cap, allowing actin monomers to bind, leading to actin polymerisation. Activation of either Rho or Rac leads to GAPs inhibiting the other

Question 30

Describe action of the Cdc42 downstream effector IQGAP.

Answer 30

Microtubules have dynamic instability, often collapsing back to the MTOC. IQGAP binds to the protein CLIP170 which is associated with the end of microtubules, stabilising them. This means microtubules that aren't at the leading edge collapse, so vesicles carrying membrane proteins and receptors travel to the leading edge. If Cdc42 is inhibited, cells fail to follow chemoattractants as receptors are not delivered to filopodia so polarisation does not occur