Molecular Analysis of SNAP-25 Function in Exocytosis GRAHAM, MARGARET E.; WASHBOURNE, PHILIP; WILSON, MICHAEL C. ...
Annals of the New York Academy of Sciences,
October 2002, Volume:
971, Issue:
1
Journal Article
Peer reviewed
: It is generally accepted that the SNARE proteins form the core of the machinery for intracellular membrane fusion and that formation of a SNARE complex is crucially important. Our aim is to dissect ...the molecular roles of the SNARE proteins and their regulators in physiological membrane fusion during exocytosis. We have developed approaches that allow us to manipulate protein expression in model secretory cells, PC12 and adrenal chromaffin cells, and to combine this with assay of exocytosis at high‐time resolution using carbon‐fiber amperometry. This technique allows us to assess the extent of exocytosis and to follow the kinetics of single secretory granule release events with millisecond time resolution. We established that manipulation of proteins involved in the exocytotic machinery can lead to detectable and interpretable changes in exocytosis kinetics that have revealed novel roles in late stages of exocytosis. Using this approach we have begun to analyze the function of SNAP‐25B using a mutant resistant to the Clostridial neurotoxin BoNT/E. This SNAP‐25 mutant can reconstitute exocytosis in BoNT/E‐treated cells. With this construct it is possible to analyze the consequences of any introduced mutation in the absence of functional endogenous protein. We review here its use in the analysis of palmitoylated cysteines of SNAP‐25 and the conserved residues of the 0 layer of the SNARE complex. The data suggest an important role of the cysteines, but not the 0 layer glutamines, in triggered exocytosis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
J. Neurochem. (2011) 117, 765–778.
Recent studies have identified the leucine rich repeat protein LRRTM2 as a post‐synaptic ligand of Neurexins. Neurexins also bind the post‐synaptic adhesion ...molecules, Neuroligins. All three families of genes have been implicated in the etiologies of neurodevelopmental disorders, specifically autism spectrum disorders and schizophrenia. Does the binding promiscuity of Neurexins now suggest complex cooperativity or redundancy at the synapse? While recent studies in primary neuronal cultures and also systematic extracellular protein interaction screens suggest summative effects of these systems, we propose that studying these interactions in the developing zebrafish embryo or larvae may shed more light on their functions during synaptogenesis in vivo. These gene families have recently been extensively characterized in zebrafish, demonstrating high sequence conservation with the human genes. The simpler circuitry of the zebrafish, together with the characterization of the expression patterns down to single, identifiable neurons and the ability to knock‐down or over‐express multiple genes in a rapid way lend themselves to dissecting complex interaction pathways. Furthermore, the capability of performing high‐throughput drug screens suggests that these small vertebrates may prove extremely useful in identifying pharmacological approaches to treating autism spectrum disorders.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell ...adhesion molecules 1-3 (SynCAM1-3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1-3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1-3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1-3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1-3 KD neurons in the same culture demonstrate that SynCAM1-3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1-3 has concealed their synaptogenic function at the postsynaptic terminal.
Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. ...Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Techniques for gene transfer into neurons Washbourne, Philip; McAllister, A.Kimberley
Current Opinion in Neurobiology,
10/2002, Volume:
12, Issue:
5
Book Review, Journal Article
Peer reviewed
To illuminate the function of the thousands of genes that make up the complexity of the nervous system, it is critical to be able to introduce and express DNA in neurons. Over the past two decades, ...many gene transfer methods have been developed, including viral vectors, liposomes and electroporation. Although the perfect gene transfer technique for every application has not yet been developed, recent technical advances have facilitated the ease of neuronal gene transfer and have increased the accessibility of these techniques to all laboratories. In order to select a transfection method for any particular experiment, the specific advantages and disadvantages of each technique must be considered.
Recent advances in transfection technologies, using both viral and non-viral methods, are making it possible to address the functions of proteins in neuronal development and adulthood in new and exciting ways.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute ...brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults.
Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages.
We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here ...we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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