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

Abstract

SummaryThe 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.

Self-organization of protrusions and polarity during eukaryotic chemotaxis

“…This behavior is also observed for other actin nucleation-promoting factors: N-WASP recycles faster in the presence of Arp2/3 complex binding and actin polymerization [48]. While the specific mechanisms mediating negative feedback have yet to be identified, recent evidence using XTC cells suggests that actin filaments may mechanically remove WRC from the membrane while undergoing retrograde flow [23] (Figure 1B). Actin may also potentially play an indirect role via the recruitment of actin-binding proteins to the leading edge that promote dissociation of WRC from the plasma membrane.…”

“…The actin cytoskeleton in motile cells is also intrinsically excitable, but in a manner that may not be dependent on upstream signals such as Ras and PIP3 [10]. While multiple feedback loops do appear to exist both within and between the signaling network and cytoskeleton [20,23–27] (Figure 1B), the specific roles of each connection in modulating cell migration are not yet clear. In its most basic form, an excitable system comprises two components, one which produces fast-acting positive feedback and a second which produces slower negative feedback [28].…”

“…Actin filaments appear to play a prominent role, as treating motile cells with drugs blocking actin assembly increases the lifetimes and intensities of WRC waves, whereas treatment with drugs stabilizing filaments has the opposite effect [10,11,23]. This behavior is also observed for other actin nucleation-promoting factors: N-WASP recycles faster in the presence of Arp2/3 complex binding and actin polymerization [48].…”

Direct interaction between exocyst and Wave complexes promotes cell protrusions and motility

“…As originally reported using a single-molecule imaging approach (Millius et al, 2012), we also observed rare events of WRC retrograde motion, which we manually tracked to find a median speed of 0.161 µm/s (s.d.=0.036, n=13 dots from five cells); this is consistent with the notion that a portion of the WRC population is recycled through incorporation into the actin network, undergoing retrograde flow (Millius et al, 2012). However, most of the movements of these Abi-positive dots at protrusions were not (upper panel) and the detection of Sec6, Sec8, Sec5, Exo70 and Exo84 (middle panel).…”

Model of turnover kinetics in the lamellipodium: implications of slow- and fast- diffusing capping protein and Arp2/3 complex

“…Slowly-diffusing capping proteins have been observed by SiMS microscopy [15], which may reflect capping protein bound to slowly-diffusing actin oligomers or to the membrane. The Arp2/3 complex has also been observed to form a slowly-diffusing complex with its activators prior to attachment to the actin network [16]. However the implications of slowly-diffusing capping protein and Arp2/3 complex on the spatial distribution and turnover kinetics in the lamellipodium has not been modeled.…”