No abstract

Despite the wealth of different actin structures formed, only two actin nucleation factors are well established in vertebrates: the Arp2/3 complex and formins. Here, we describe a further nucleator, cordon-bleu (Cobl). Cobl is a brain-enriched protein using three Wiskott-Aldrich syndrome protein homology 2 (WH2) domains for actin binding. Cobl promotes nonbundled, unbranched filaments. Filament formation relies on barbed-end growth and requires all three Cobl WH2 domains and the extended linker L2. We suggest that the nucleation power of Cobl is based on the assembly of three actin monomers in cross-filament orientation. Cobl localizes to sites of high actin dynamics and modulates cell morphology. In neurons, induction of both neurites and neurite branching is dramatically increased by Cobl expression-effects that critically depend on Cobl's actin nucleation ability. Correspondingly, Cobl depletion results in decreased dendritic arborization. Thus, Cobl is an actin nucleator controlling neuronal morphology and development.

Against the odds of membrane resistance, members of the BIN/Amphiphysin/Rvs (BAR) domain superfamily shape membranes and their activity is indispensable for a plethora of life functions. While crystal structures of different BAR dimers advanced our understanding of membrane shaping by scaffolding and hydrophobic insertion mechanisms considerably, especially life-imaging techniques and loss-of-function studies of clathrinmediated endocytosis with its gradually increasing curvature show that the initial idea that solely BAR domain curvatures determine their functions is oversimplified. Diagonal placing, lateral lipid-binding modes, additional lipid-binding modules, tilde shapes and formation of macromolecular lattices with different modes of organisation and arrangement increase versatility. A picture emerges, in which BAR domain proteins create macromolecular platforms, that recruit and connect different binding partners and ensure the connection and coordination of the different events during the endocytic process, such as membrane invagination, coat formation, actin nucleation, vesicle size control, fission, detachment and uncoating, in time and space, and may thereby offer mechanistic explanations for how coordination, directionality and effectiveness of a complex process with several steps and key players can be achieved.

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.