#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

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 micro

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. F

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 ex

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

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