Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers1. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect2–4, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin5,6. Clathrin-independent endocytic events are often less reliant on dynamin7, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we found that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which both are clathrin-independent endocytic cargoes8. In controlled in vitro systems, endoA2 reshapes membranes prior to scission. Furthermore, we demonstrate that endoA2, dynamin, and actin contribute in parallel to the scission of Shiga toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings finally highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling force-driven dynamic scission.

Abstract. Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. G6raud, A. Santa Maria, B. Maro, V. Mayan, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type tab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Overexpression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (ot-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

Synaptic vesicles, which have been a paradigm for the fusion of a vesicle with its target membrane, also serve as a model for understanding the formation of a vesicle from its donor membrane. Synaptic vesicles, which are formed and recycled at the periphery of the neuron, contain a highly restricted set of neuronal proteins. Insight into the trafficking of synaptic vesicle proteins has come from studying not only neurons but also neuroendocrine cells, which form synaptic-like microvesicles (SLMVs). Formation and recycling of synaptic vesicles/SLMVs takes place from the early endosome and the plasma membrane. The cytoplasmic machinery of synaptic vesicle/SLMV formation and recycling has been studied by a variety of experimental approaches, in particular using cell-free systems. This has revealed distinct machineries for membrane budding and fission. Budding is mediated by clathrin and clathrin adaptors, whereas fission is mediated by dynamin and its interacting protein SH3p4, a lysophosphatidic acid acyl transferase.

The development of oxytocin (OT) receptors in the rat brain and spinal cord was studied by in vitro light microscopic autoradiography and by electrophysiology. OT receptors were labeled using a monoiodinated OT antagonist in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the response of ongoing spike activity to the addition of OT was assessed in neurons located in the dorsal motor nucleus of the vagus nerve of the neonate. Specific binding was detected first at E14 in a region that later differentiated into the dorsal motor nucleus of the vagus nerve. Many other regions were progressively labeled between E20 and PN5. From PN5 to PN16, the distribution of binding sites remained essentially unchanged but differed markedly from that characteristic of the adult. The change-over from the "infant pattern" to the "adult pattern" occurred in 2 stages: the first change took place between PN16 and PN22, a time corresponding to the preweaning period; the second change occurred after PN35 and thus coincided with the onset of puberty. During the first transition period, binding was reduced or disappeared in several areas intensely labeled at earlier stages, in particular, in the cingulate cortex and the dorsal hippocampus. At the same time, binding sites appeared in the ventral hippocampus. At puberty, high densities of OT binding sites appeared in the ventromedial hypothalamic nucleus and the olfactory tubercle. Electrophysiological activity was recorded from vagal neurons in slices obtained from animals sacrificed at PN1-PN12. OT and a selective OT agonist reversibly increased the firing rate of these neurons in a concentration-dependent manner. The neuronal responsiveness was similar to that reported previously in the adult. These results suggest that OT binding sites detected by autoradiography in the developing rat brain represent, at least in some areas, functional neuronal receptors.

This first-in-human study of RTH258 demonstrated noninferiority in the change in CSFT at 1 month for the 4.5- and 6.0-mg doses compared with ranibizumab and an increase of 30 days in the median time to PBT for the 6.0-mg dose. There were no unexpected safety concerns, and the results support the continued development of RTH258 for the treatment of neovascular AMD.

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

The BKH-N provides national data based on the notification of OSDs in Germany. The results demonstrate the rank of occupations hazardous for the skin and are helpful for defining target groups for prevention.