Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Big gulps: specialized membrane domains for rapid receptor-mediated endocytosis

Eukaryotic cells are well known to use an elaborate, clathrin-based endocytic machinery to internalize ligand-receptor complexes. Although the number of components identified as being used during this essential process has increased substantially over the past decade, an appreciation for how receptor-mediated endocytosis is organized at a broader, cellular scale is still mostly undefined. Here, some new insights into how cells cluster and organize the endocytic machinery at defined cytoplasmic domains to increase the speed and efficiency of ligand-receptor internalization are presented.     

Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells

The bacterial pathogen Listeria monocytogenes uses the surface protein InlB to invade a variety of cell types. The interaction of InlB with the hepatocyte growth-factor receptor, Met, is crucial for infection to occur. Remarkably, the ubiquitin ligase Cbl is rapidly recruited to InlB-activated Met. Recent studies have shown that ligand-dependent endocytosis of Met and other receptor tyrosine kinases is triggered by monoubiquitination of the receptor, a process that is mediated by Cbl. Here, we show that purified InlB induces the Cbl-dependent monoubiquitination and endocytosis of Met. We then demonstrate that the bacterium exploits the ubiquitin-dependent endocytosis machinery to invade mammalian cells. First, we show that L. monocytogenes colocalizes with Met, EEA1, Cbl, clathrin and dynamin during entry. Then, we assess the role of different proteins of the endocytic machinery during L. monocytogenes infection. Over-expression or down-regulation of Cbl, respectively, increases or decreases bacterial invasion. Furthermore, RNA interference-mediated knock-down of major components of the endocytic machinery (for example, clathrin, dynamin, eps15, Grb2, CIN85, CD2AP, cortactin and Hrs), inhibit bacterial entry, establishing that the endocytic machinery is key to the bacterial internalization process.     

The endocytic machinery in nerve terminals surrounds sites of exocytosis

In most models of endocytosis, the endocytic machinery is recruited from the cytoplasm by cytoplasmic tails of the plasma membrane proteins that are to be internalized. This does not appear to be true at synapses where the endocytic machinery required for synaptic vesicle recycling is localized to membrane-associated 'hot spots' [1] [2]. In Drosophila neuromuscular junctions, the multi-domain protein Dap160 is also localized to hot spots [3] and has some characteristics expected of an anchoring protein. Anchoring the endocytic machinery to the plasma membrane might help contribute to the remarkable speed of synaptic vesicle recycling [4]. Here, we report that the endocytic machinery surrounds sites that are believed to be sites of exocytosis. We propose that the radial distribution of the synaptic vesicle recycling machinery already present on the plasma membrane in unstimulated nerve terminals is a fundamental unit of pre-synaptic organization and allows the nerve terminal to extract maximum recycling efficiency out of conventional endocytic machinery.     

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.     

Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.     

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.     

Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 +/- 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.     

Oncogenic breakdowns in endocytic adaptor proteins

Endocytosis is a versatile tool to regulate the intensity, localization, half-life and function of signaling complexes (signalosomes) that form in cells upon binding of growth factors, cytokines and morphogens to their cognate receptors. Endocytic adaptors are non-catalytic proteins that assemble effectors and structural components of the endocytic machinery around the trafficking cargo and serve as scaffolds for signalosomes, which in turn modify their location and activity by various post-translational modifications. We discuss how breakdowns in the function of endocytic adaptors might facilitate impairment of tissue homeostasis and consequent tumor development.     

The regulation of endocytosis: identifying dynamin's binding partners

Dynamin is a large GTPase implicated in controlling the initial stages of endocytosis. Its mechanism of action remains uncertain, but it is expected to interact cyclically with components of the endocytic machinery. Dynamin binds to a number of different macromolecules, including microtubules, SH3-domain-containing proteins, and acidic phospholipids. We interpret these findings in terms of a cooperative interaction between dynamin and components of coated and non-coated pits.     

Endocytic proteins: An expanding repertoire of presynaptic functions

From a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca²⁺-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Structural insights into clathrin-mediated endocytosis

The process of clathrin-mediated endocytosis from the plasma membrane has been the subject of many biological and biochemical investigations. Recent atomic resolution structures determined by X-ray crystallography now enable the molecular basis for the interactions of some components of the endocytic machinery to be understood in detail.     

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. Here, we provide evidence for a clathrin-independent endocytic pathway in yeast. In cells lacking the clathrin-binding adaptor proteins Ent1, Ent2, Yap1801, and Yap1802, we identify a second endocytic pathway that depends on the GTPase Rho1, the downstream formin Bni1, and the Bni1 cofactors Bud6 and Spa2. This second pathway does not require components of the better-studied endocytic pathway, including clathrin and Arp2/3 complex activators. Thus, our results reveal the existence of a second pathway for endocytosis in yeast, which suggests similarities with the RhoA-dependent endocytic pathways of mammalian cells.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Evolving endosomes: how many varieties and why?

The cell biologist's insight into endosomal diversity, in terms of both form and function, has increased dramatically in the past few years. This understanding has been promoted by the availability of powerful new techniques that allow imaging of both cargo and machinery in the endocytic process in real time, and by our ability to inhibit components of this machinery by RNA interference. The emerging picture from these studies is of a highly complex, dynamic and adaptable endosomal system that interacts at various points with the secretory system of the cell.     

A novel AP-2 adaptor interaction motif initially identified in the long-splice isoform of synaptojanin 1, SJ170

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded alpha-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.     

The endocytic machinery at an interface with the actin cytoskeleton: a dynamic,hip intersection

Clathrin-mediated endocytosis is the major mechanism by which proteins and membrane lipids gain access into cells. Over the past several years, an array of proteins has been identified that define the molecular machinery regulating the formation of clathrin-coated pits and vesicles. This article focuses on how the identification of this machinery has begun to reveal a molecular basis for a link between endocytosis and the actin cytoskeleton--a link that had long been suspected to exist in mammalian cells but which had remained elusive. In particular, I discuss the relationship between actin and three components of the endocytic machinery--dynamin, HIPs (huntingtin-interacting proteins) and intersectin.     

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium

Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.     

p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.