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Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.

Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)- trisphosphate (PIP3) production decreases CIP4 at themembrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge.We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons. [ABSTRACT FROM AUTHOR]

Summary: Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing membrane curvature []. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types []. However, there is little data on how CIP4 functions in neurons []. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal versus nonneuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures. [Copyright &y& Elsevier]