Project description:The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.
Project description:Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (Ca(V)) channels. Here we show that the functional expression of neuronal N-type Ca(V) channels (Ca(V)2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases Ca(V) channel density in somata and in presynaptic terminals. We then show that FMRP controls Ca(V)2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and Ca(V)2.2 occurs between the carboxy-terminal domain of FMRP and domains of Ca(V)2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via Ca(V)2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.
Project description:A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.
Project description:Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.
Project description:Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.
Project description:Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.
Project description:Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.
Project description:The rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. Here, we show that posttranslational modification of the active zone protein Rab3-interacting molecule 1? (RIM1?) by the small ubiquitin-like modifier 1 (SUMO-1) functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1? SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca(2+) influx necessary for vesicular release, whereas non-SUMOylated RIM1? participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1? is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated "switching" of RIM1? between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction.
Project description:Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II-dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.
Project description:Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.