Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation ...of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis.
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•Synaptojanin and endophilin accelerate ultrafast endocytosis•Neck formation requires endophilin and the 5-phosphatase activity of synaptojanin•Synaptic vesicles still form from synaptic endosomes in absence of these proteins•Endophilin and 4- and 5-phosphatase are required for clathrin uncoating
Ultrafast endocytosis is a distinct form of synaptic vesicle recovery occurring within milliseconds of vesicle fusion. Using flash-and-freeze electron microscopy, Watanabe et al. demonstrate dual roles for synaptojanin and endophilin in membrane remodeling and clathrin uncoating on rapid timescales during ultrafast endocytosis.
Plant Golgi ultrastructure ROBINSON, DAVID G.
Journal of microscopy (Oxford),
November 2020, Letnik:
280, Številka:
2
Journal Article
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Summary
The plant Golgi apparatus (sensu lato: Golgi stack + Trans Golgi Network, TGN) is a highly polar and mobile key organelle lying at the junction of the secretory and endocytic pathways. Unlike ...its counterpart in animal cells it does not disassemble during mitosis. It modifies glycoproteins sent to it from the endoplasmic reticulum (ER), it recycles ER resident proteins, it sorts proteins destined for the vacuole from secretory proteins, it receives proteins internalised from the plasma membrane and either recycles them to the plasma membrane or retargets them to the vacuole for degradation. In functional terms the Golgi apparatus can be likened to a car factory, with incoming (COPII traffic) and returning (COPI traffic) railway lines at the entry gate, and a distribution centre (the TGN) at the exit gate of the assembly hall. In the assembly hall we have a conveyor belt system where the incoming car parts are initially assembled (in the cis‐area) then gradually modified into different models (processing of secretory cargo) as the cars pass along the production line (cisternal maturation). After being released the trans‐area, the cars (secretory cargos) are moved out of the assembly hall and passed on to the distribution centre (TGN), where the various models are placed onto different trains (cargo sorting into carrier vesicles) for transport to the car dealers. Cars with motor problems are returned to the factory for repairs (endocytosis to the TGN). This simple analogy also incorporates features of quality control at the COPII entry gate with defective parts being returned to the manufacturing center (the ER) via the COPI trains (vesicles).
In recent years, numerous studies have contributed to our knowledge on Golgi function and structure in both animals, yeast and plants. This review, rather than giving a balanced account of the structure as well as of the function of the Golgi apparatus has purposely a marked slant towards plant Golgi ultrastructure integrating findings from the mammalian/animal field.
The protein α‐synuclein has a central role in the pathogenesis of Parkinson’s disease (PD). In this review, we discuss recent results concerning its primary function, which appears to be on cell ...membranes. The pre‐synaptic location of synuclein has suggested a role in neurotransmitter release and it apparently associates with synaptic vesicles because of their high curvature. Indeed, synuclein over‐expression inhibits synaptic vesicle exocytosis. However, loss of synuclein has not yet been shown to have a major effect on synaptic transmission. Consistent with work showing that synuclein can promote as well as sense membrane curvature, recent analysis of synuclein triple knockout mice now shows that synuclein accelerates dilation of the exocytic fusion pore. This form of regulation affects primarily the release of slowly discharged lumenal cargo such as neural peptides, but presumably also contributes to maintenance of the release site.
This article is part of the Special Issue “Synuclein”.
This review addresses the physiological role of alpha‐synuclein from experiments on the behavior of synuclein in vitro to the imaging of regulated exocytosis in live neurons from knockout mice. Over‐expression of synuclein has been shown to inhibit regulated exocytosis, but loss of synuclein has little effect on membrane fusion. Rather, the loss of all three synuclein genes impairs dilation of the fusion pore that forms during exocytosis. The mechanism by which synuclein promotes vesicle collapse remains unknown.
This article is part of the Special Issue “Synuclein”.
The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. Here we use tetanus neurotoxin (TeNT) light chain, ...which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. Expression of tetanus insensitive (TI)-VAMP2 restored SV fusion in the presence of TeNT, but not DCV fusion. Expression of TI-VAMP1 or TI-VAMP3 also failed to restore DCV fusion. Co-transport assays revealed that both TI-VAMP1 and TI-VAMP2 are targeted to DCVs and travel together with DCVs in neurons. Furthermore, expression of the TeNT-cleaved VAMP2 fragment or a protease defective TeNT in wild type neurons did not affect DCV fusion and therefore cannot explain the lack of rescue of DCV fusion by TI-VAMP2. Finally, to test if two different VAMPs might both be required in the DCV secretory pathway, Vamp1 null mutants were tested. However, VAMP1 deficiency did not reduce DCV fusion. In conclusion, TeNT treatment combined with TI-VAMP2 expression differentially affects the two main regulated secretory pathways: while SV fusion is normal, DCV fusion is absent.
Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but ...the role of syp in the process of membrane fusion during Ca
-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.
The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca
-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.
Membrane vesicles are secreted by growing bacterial cells and are important components of the bacterial secretome, with a role in delivering effector molecules that ultimately enable bacterial ...survival. Membrane vesicles of Clostridioides difficile likely contribute to pathogenicity and is a new area of research on which there is currently very limited information. This chapter summarizes the current knowledge on membrane vesicle formation, content, methods of characterization and functions in Clostridia and model Gram-positive species.
Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences ...co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.
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•Electron tomography enables the dissection of vesicle pools at synaptic active zones•Docked and primed vesicle availability contributes to initial release probability•The ratio of docked and tethered vesicles may co-determine short-term plasticity•Hippocampal mossy fibers contain three morphological types of docked vesicles
Distinct synapse types exhibit strikingly different morphological and functional properties. To investigate how the ultrastructural architecture of synaptic release sites contributes to such diversity, Maus et al. exploit electron tomography to correlate the nanoscale organization of synaptic vesicle pools with fundamental functional properties such as short-term synaptic plasticity characteristics.
Efficient neurotransmitter release at the presynaptic terminal requires docking of synaptic vesicles to the active zone membrane and formation of fusion-competent synaptic vesicles near voltage-gated ...Ca
channels. Rab3-interacting molecule (RIM) is a critical active zone organizer, as it recruits Ca
channels and activates synaptic vesicle docking and priming via Munc13-1. However, our knowledge about Munc13-independent contributions of RIM to active zone functions is limited. To identify the functions that are solely mediated by RIM, we used genetic manipulations to control RIM and Munc13-1 activity in cultured hippocampal neurons from mice of either sex and compared synaptic ultrastructure and neurotransmission. We found that RIM modulates synaptic vesicle localization in the proximity of the active zone membrane independent of Munc13-1. In another step, both RIM and Munc13 mediate synaptic vesicle docking and priming. In addition, while the activity of both RIM and Munc13-1 is required for Ca
-evoked release, RIM uniquely controls neurotransmitter release efficiency. However, activity-dependent augmentation of synaptic vesicle pool size relies exclusively on the action of Munc13s. Based on our results, we extend previous findings and propose a refined model in which RIM and Munc13-1 act in overlapping and independent stages of synaptic vesicle localization and release.
The presynaptic active zone is composed of scaffolding proteins that functionally interact to localize synaptic vesicles to release sites, ensuring neurotransmission. Our current knowledge of the presynaptic active zone function relies on structure-function analysis, which has provided detailed information on the network of interactions and the impact of active zone proteins. Yet, the hierarchical, redundant, or independent cooperation of each active zone protein to synapse functions is not fully understood. Rab3-interacting molecule and Munc13 are the two key functionally interacting active zone proteins. Here, we dissected the distinct actions of Rab3-interacting molecule and Munc13-1 from both ultrastructural and physiological aspects. Our findings provide a more detailed view of how these two presynaptic proteins orchestrate their functions to achieve synaptic transmission.
Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid, and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly ...understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein vesicle associated protein associated protein A (VAP‐A) drives biogenesis of a subset of RNA‐enriched EVs. Here, we examine the protein content of VAP‐A‐regulated EVs. Using label‐free proteomics, we identified down‐ and upregulated proteins in small EVs (SEVs) purified from VAP‐A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP‐A. Search Tool for the Retrieval of Reciprocity Genes (STRING) protein–protein interaction network analysis of the RNA‐binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP‐A KD SEVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin‐binding, unfolded protein binding, and ATP‐dependent proteins.
A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is ...necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within < 100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.
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