Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by singlemolecule imaging

Arthur Millius, Naoki Watanabe, Orion D. Weiner

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)

Abstract

The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes.

Original languageEnglish
Pages (from-to)1165-1176
Number of pages12
JournalJournal of cell science
Volume125
Issue number5
DOIs
Publication statusPublished - 2012 Mar 1
Externally publishedYes

Keywords

  • Actin cytoskeleton
  • Diffusion
  • Retrograde flow
  • SCAR/WAVE complex
  • Single-molecule imaging

ASJC Scopus subject areas

  • Cell Biology

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