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#actin nucleation

2 public questions tagged with this topic.

Cytoskeleton

What is the molecular weight of the Arp2/3 complex?

Arp2/3 complex purification and characterization resolved molecular composition and mass. Original isolation from Acanthamoeba by Pollard demonstrated seven polypeptides co fractionating through multiple chromatographic steps with actin binding activity. Subunit sequencing identified two actin related proteins Arp2 44 kilodalton and Arp3 50 kilodalton sharing actin fold, plus ARPC1 p40 WD repeat beta propeller 40 kilodalton, ARPC2 p34 34 kilodalton, ARPC3 p21 21 kilodalton, ARPC4 p20 20 kilodalton, ARPC5 p16 14 to 16 kilodalton. Sum calculated 224 kilodalton, sucrose gradient sedimentation and gel filtration elution volume around 200 to 220 kilodalton confirming monomeric assembly. Cryo electron microscopy volume consistent with 15 nanometer globular particle. Higher mass 400 to 600 kilodalton would imply dimer or association with nucleation promoting factor, lower 30 to 50 corresponds to actin monomer, 100 to 150 would be incomplete subcomplex lacking several subunits, insufficient for nucleation. Therefore assignment 200 to 220 kilodalton aligns with structural and biochemical consensus for intact active complex and functional unit.

Ref: Pollard and Earnshaw, Cell Biology, Chapter 12: Arp2/3 Complex Molecular Weight and Composition.

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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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