The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPFs). Recently, the WASH family of NPFs has been identified, but its cellular role is unclear. Here, we show that WASH generates an actin network on a restricted domain of sorting and recycling endosomes. We found that WASH belongs to a multiprotein complex containing seven subunits, including the heterodimer of capping protein (CP). In vitro, the purified WASH complex activates Arp2/3-mediated actin nucleation and binds directly to liposomes. WASH also interacts with dynamin. WASH depletion gives rise to long membrane tubules pulled out from endosomes along microtubules, as does dynamin inhibition. Accordingly, WASH is required for efficient transferrin recycling. Together, these data suggest that the WASH molecular machine, integrating CP with a NPF, controls the fission of endosomes through an interplay between the forces generated by microtubule motors and actin polymerization.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used green fluorescent proteins (GFPs) with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.