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The ability of cells to manage consequences of exogenous proteotoxicity is key to cellular homeostasis. While a plethora of well-characterised machinery aids intracellular proteostasis, mechanisms involved in the response to denaturation of extracellular proteins remain elusive. Here we show that aggregation of protein ectodomains triggers their endocytosis via a macroendocytic route, and subsequent lysosomal degradation. Using ERBB2/HER2-specific antibodies we reveal that their cross-linking ability triggers specific and fast endocytosis of the receptor, independent of clathrin and dynamin. Upon aggregation, canonical clathrin-dependent cargoes are redirected into the aggregation-dependent endocytosis (ADE) pathway. ADE is an actin-driven process, which morphologically resembles macropinocytosis. Physical and chemical stress-induced aggregation of surface proteins also triggers ADE, facilitating their degradation in the lysosome. This study pinpoints aggregation of extracellular domains as a trigger for rapid uptake and lysosomal clearance which besides its proteostatic function has potential implications for the uptake of pathological protein aggregates and antibody-based therapies. How cells respond to denaturation of extracellular protein domains remained largely unknown. Here, authors describe an aggregation-dependent endocytosis pathway, facilitating uptake and degradation of antibody- and stress-induced protein aggregates.

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α 2a - and β 1 -adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME). This study describes a fast, clathrin-independent endocytic pathway mediated by endophilin, dynamin and actin; the pathway is activated by ligand binding to a variety of cargo receptors, and endophilin-mediated endocytosis occurs primarily at the leading edges of cells where lamellipodin and the lipid PtdIns(3,4)P 2 ensure endophilin targeting. Endocytosis and cell signalling Cells internalize nutrients and turnover membrane components through the process of endocytosis, which in most cases involves the protein clathrin. Endophilin has been thought to be a component of clathrin-mediated endocytosis, but two studies published in this issue of Nature show that this protein mediates a fast-acting, clathrin-independent form of endocytosis which involves formation of tubular vesicles. Emmanuel Boucrot et al . report that this pathway is triggered by binding of ligands to cargo receptors, and requires the proteins dynamin and actin. Endophilin-mediated endocytosis also seems to have distinct cellular homes, occurring at the leading edges of cells where the lipid PtdIns(3,4)P 2 ensures endophilin engagement. This form of endocytosis is shown to mediate the uptake of several physiological and disease-relevant receptors including G-protein-coupled receptors and receptor tyrosine kinases. In the second paper, Henri-François Renard et al . provide evidence that bacterial toxins take advantage of the same pathway to enter cells, and also find that endophilin-A2 acts together with dynamin and actin.

Clathrin-mediated endocytosis, the major pathway for ligand internalization into eukaryotic cells, is thought to be initiated by the clustering of clathrin and adaptors around receptors destined for internalization. However, here we report that the membrane-sculpting F-BAR domain-containing Fer/Cip4 homology domain-only proteins 1 and 2 (FCHo1/2) were required for plasma membrane clathrin-coated vesicle (CCV) budding and marked sites of CCV formation. Changes in FCHo1/2 expression levels correlated directly with numbers of CCV budding events, ligand endocytosis, and synaptic vesicle marker recycling. FCHo1/2 proteins bound specifically to the plasma membrane and recruited the scaffold proteins eps15 and intersectin, which in turn engaged the adaptor complex AP2. The FCHo F-BAR membrane-bending activity was required, leading to the proposal that FCHo1/2 sculpt the initial bud site and recruit the clathrin machinery for CCV formation.

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

The ubiquitous Ca 2+ sensor calmodulin (CaM) binds and regulates many proteins, including ion channels, CaM kinases, and calcineurin, according to Ca 2+ -CaM levels. What regulates neuronal CaM levels, is, however, unclear. CaM-binding transcription activators (CAMTAs) are ancient proteins expressed broadly in nervous systems and whose loss confers pleiotropic behavioral defects in flies, mice, and humans. Using Caenorhabditis elegans and Drosophila , we show that CAMTAs control neuronal CaM levels. The behavioral and neuronal Ca 2+ signaling defects in mutants lacking camt-1, the sole C. elegans CAMTA, can be rescued by supplementing neuronal CaM. CAMT-1 binds multiple sites in the CaM promoter and deleting these sites phenocopies camt-1 . Our data suggest CAMTAs mediate a conserved and general mechanism that controls neuronal CaM levels, thereby regulating Ca 2+ signaling, physiology, and behavior.

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P 2 in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P 2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

Cell motility, adhesion and phagocytosis are controlled by actin and membrane remodelling processes. Bridging integrator-2 (Bin2) also called Breast cancer-associated protein 1 (BRAP1) is a predicted N-BAR domain containing protein with unknown function that is highly expressed in leucocytic cells. In the present study we solved the structure of Bin2 BAR domain and studied its membrane binding and bending properties in vitro and in vivo. Live-cell imaging experiments showed that Bin2 is associated with actin rich structures on the plasma membrane, where it was targeted through its N-BAR domain. Pull-down experiments and immunoprecipitations showed that Bin2 C-terminus bound SH3 domain containing proteins such as Endophilin A2 and α-PIX. siRNA of endogenous protein led to decreased cell migration, increased phagocytosis and reduced podosome density and dynamics. In contrast, overexpression of Bin2 led to decreased phagocytosis and increased podosome density and dynamics. We conclude that Bin2 is a membrane-sculpting protein that influences podosome formation, motility and phagocytosis in leucocytes. Further understanding of this protein may be key to understand the behaviour of leucocytes under physiological and pathological conditions.

Dynamin is a large GTPase with a relative molecular mass of 96,000 ( M r  96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes 1 , 2 . Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear 3 , 4 . It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors 5 . Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo . Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain- containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin- independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.