Kohji Takei

Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals

THE mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate1–5. Because several vesicular transport steps require GTP hydrolysis6–9, GTP-γS may help identify intermediates in synaptic vesicle recycling. In GTP-γS-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates10,11. Dynamin is a GTPase implicated in synaptic vesicle endocytosis12–14 and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.

Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Functional partnership between amphiphysin and dynamin in clathrin-mediatedendocytosis

Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.

Generation of high curvature membranes mediated by direct endophilin bilayer interactions

Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143–154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133–141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301–312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33–39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamins GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis

During endocytosis, clathrin and the clathrin adaptor protein AP-2 (ref. 1), assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane,. One of these factors is Eps15, a clathrin-coat-associated protein that binds the α-adaptin subunit of AP-2 (refs 4–8). Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 (ref. 10) and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.

GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.

GABA, a major inhibitory neurotransmitter of the brain, is also present at high concentration in pancreatic islets. Current evidence suggests that within islets GABA is secreted from beta‐cells and regulates the function of mantle cells (alpha‐ and delta‐cells). In the nervous system GABA is stored in, and secreted from, synaptic vesicles. The mechanism of GABA secretion from beta‐cells remains to be elucidated. Recently the existence of synaptic‐like microvesicles has been demonstrated in some peptide‐secreting endocrine cells. The function of these vesicles is so far unknown. The proposed paracrine action of GABA in pancreatic islets makes beta‐cells a useful model system to explore the possibility that synaptic‐like microvesicles, like synaptic vesicles, are involved in the storage and release of non‐peptide neurotransmitters. We report here the presence of synaptic‐like microvesicles in beta‐cells and in beta‐cells. Some beta‐cells in culture were found to extend neurite‐like processes. When these were present, synaptic‐like microvesicles were particularly concentrated in their distal portions. The GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), was found to be localized around synaptic‐like microvesicles. This was similar to the localization of GAD around synaptic vesicles in GABA‐secreting neurons. GABA immunoreactivity was found to be concentrated in regions of beta‐cells which were enriched in synaptic‐like microvesicles. These findings suggest that in beta‐cells synaptic‐like microvesicles are storage organelles for GABA and support the hypothesis that storage of non‐peptide signal molecules destined for secretion might be a general feature of synaptic‐like microvesicles of endocrine cells.

Generation of Coated Intermediates of Clathrin-Mediated Endocytosis on Protein-Free Liposomes

Clathrin-coated buds and dynamin-coated tubules morphologically similar to corresponding structures observed in synaptic membranes can be generated on protein-free liposomes by incubation with cytosol, or with clathrin coat proteins and purified dynamin, respectively. Dynamin- and clathrin-coated intermediates may form independently of each other, despite the coupling between the two processes typically observed in synaptic membranes. Formation of both structures on liposomes can occur in the absence of nucleotides. These findings indicate that interfaces between lipids and cytosolic proteins are fully sufficient to deform lipids bilayers into buds and tubules. They suggest that a main function of membrane proteins is to act as positive and negative regulators of coat assembly, therefore controlling these processes in time and space.

CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPγS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Iγ (PIPKIγ), in this effect. These data suggest a model according to which activation of PIPKIγ by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Clathrin-mediated endocytosis: membrane factors pull the trigger

Clathrin-mediated endocytosis is a vesicular transport event involved in the internalization and recycling of receptors participating in signal transduction events and nutrient import as well as in the reformation of synaptic vesicles. Recent studies in vitro and in living cells have provided a number of new insights into the initial steps of clathrin-coated vesicle formation and the membrane factors involved in this process. The unexpected complexity of these interactions at the cytosol-membrane interface suggests that clathrin-coated vesicle assembly is a highly cooperative process occurring under tight regulatory control. In this review, we focus on the role of membrane proteins and lipids in the nucleation of clathrin-coated pits and provide a hypothetical model for the early steps in clathrin-mediated endocytosis.