#actin assembly

Cytoskeleton

In receptor-mediated endocytosis, which protein activates the Arp2/3 complex to drive actin assembly?

Endocytosis of cargo such as EGFR transferrin requires actin assistance in mammalian cells against high membrane tension especially under high membrane stiffness. Clathrin coated pit maturation involves transient actin patch assembly lasting 10 to 20 seconds around neck. Key signaling axis Cdc42 GTP recruits N WASP, which in resting state autoinhibited by intramolecular interaction between basic region GBD and VCA. Activation occurs when Cdc42 GTP binds GBD, PIP2 binds basic region, and SH3 proteins TOCA1 syndapin bind proline rich stretch, releasing VCA. Free VCA binds one G actin via WH2 and Arp2/3 via acidic region bringing complex to side of preexisting filament. Branch nucleation at 70 degrees generates pushing force assisting invagination and cooperates with dynamin GTPase for scission. Cells from Wiskott Aldrich syndrome patients lacking functional WASP show defective TCR endocytosis and actin foci. Formins create unbranched filaments elsewhere, myosin I provides tension but not activation, CapZ restricts growth. Thus WASP family as WASp functions as initiator activating Arp2/3 to drive actin assembly during receptor mediated endocytosis and vesicle formation.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 13: WASP Arp2/3 Activation in Endocytosis.

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Cytoskeleton

Which of the following proteins acts as a nucleation promoting factor for the Arp2/3 complex?

Arp2/3 mediated branch formation depends on nucleation promoting factors bearing C central A acidic region plus WH2 actin binding motifs. Endogenous factors include N WASP activated by Cdc42 and PIP2 in endocytosis and filopodia, WAVE activated by Rac at lamellipodia, WHAMM at Golgi. Bacterial pathogens mimic motif to hijack host cytoskeleton. ActA surface protein of Listeria monocytogenes 639 amino acids contains N terminal polycationic stretch, central proline rich repeats binding Ena VASP and acidic actin binding motifs functioning as VCA equivalent recruiting Arp2/3 with affinity 0.3 micromolar. By polar localization achieved through Sec translocation and proteolytic processing leaving ActA concentrated at old pole, bacterium creates sustained local activation generating polarized comet tail propulsion at 0.05 to 0.3 micron per second enabling cell to cell spread without extracellular exposure. Rho GTP activates formin ROCK but not directly Arp2/3 as NPF, gelsolin caps and severs, vinculin links adhesion plaques. Thus ActA exemplary bacterial NPF for Arp2/3 dependent motility and spread.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: ActA as NPF for Arp2/3 Activation.

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Cytoskeleton

What is the role of FH2 domain in formin proteins?

Formin FH domains orchestrate assembly of long unbranched filaments in filopodia, dorsal stress fibers, yeast actin cables and contractile rings. FH2 domain 400 amino acids forms head to tail dimer with two actin binding sites, donut shaped ring encircling barbed end. Structural snapshots in different nucleotide states support stair stepping model where dimer steps onto newly added subunit without dissociating, remaining processively attached enabling continuous insertion while preventing CapZ binding. FH2 alone sufficient for nucleation in vitro, albeit slow, FH1 domain enhances efficiency. FH1 contains 10 to 15 polyproline motifs that bind profilin ATP actin via hydrophobic pocket, delivering monomers to barbed end at high local concentration, accelerating elongation up to 15 fold. This leaky capping behavior distinguishes formins from pure cappers. Thymosin beta4 sequesters monomers blocking both ends, profilin alone does not nucleate, FH2 does not recruit Arp2/3 machinery. Thus nucleation and persistent barbed association defines FH2 functionality and processive elongation mechanism.

Ref: Pollard and Earnshaw, Cell Biology, Chapter 12: Formin FH2 Domain Nucleation and Processivity.

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Cytoskeleton

Which of the following conditions favors F-actin polymerization?

F actin formation prerequisites can be recapitulated in vitro using purified muscle actin from rabbit skeletal muscle. G actin in Ca ATP state low ionic strength is monomeric. Initiation requires exchange of Ca2+ for Mg2+ at high affinity site by adding 2 millimolar MgCl2 and 50 to 100 millimolar KCl to screen electrostatic repulsion, plus 1 millimolar ATP to maintain ATP actin. Crucially thermodynamic requirement total G actin concentration must exceed critical concentration about 0.1 micromolar at plus end. Below this no polymer forms, above this equilibrium free monomer remains at Cc and excess converts to filament, detected by increase in pyrene fluorescence, light scattering or sedimentation. High cofilin raises effective Cc by increasing off rate and severing, low salt prevents nucleation by failing to neutralize surface charges, absence ATP yields ADP actin with higher Cc and unstable filaments. Experiments routinely use 5 micromolar actin in F buffer containing MgCl2 KCl ATP, often adding spectrin actin seeds or formin FH2 to bypass slow nucleation phase, ensuring rapid polymerization for motility assays and structural studies.

Ref: Pollard and Earnshaw, Cell Biology, Chapter 12: Conditions for F-Actin Polymerization and Cc.

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