Nanoscale organization of the pre-synapse: Tracking the neurotransmitter release machinery

Chemical communication is underpinned by the fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane at active zones. With the advent of super-resolution microscopy, the door is now opened to unravel the dynamic remodeling of synapses underpinning learning and memory. Imaging proteins with conventional light microscopy cannot provide submicron information vital to determining the nanoscale organization of the synapse. We will first review the current super-resolution microscopy techniques available to investigate the localization and movement of synaptic proteins and how they have been applied to visualize the synapse. We discuss the new techniques and analytical approaches have provided comprehensive insights into synaptic organization in various model systems. Finally, this review provides a brief update on how these super-resolution techniques and analyses have opened the way to a much greater understanding of the synapse, the fusion and compensatory endocytosis machinery.     

A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa

Antibodies to the Saccharomyces cereviseae plasma membrane t-SNARE Sso2p identify a putative 39-kDa homologue in Neurospora crassa. The 39-kDa protein is enriched in plasma membrane (PM) and occurred with other membranes. It is extractable by detergent, but not chaotropic or alkali agents, suggesting membrane insertion. Immunoprecipitation with anti-Sso2p coprecipitated a approximately 100-kDa, Mg(+)-ATP-sensitive band with the 39-kDa protein, suggesting a ternary SNARE complex. Affinity-purified anti-Sso2p gave hyphal staining patterns most consistent with protein localization on both the PM and intracellular exocytotic apical wall vesicles. The PM staining in hyphal apices formed a tip-high gradient, not as steep as that predicted by the "hyphoid equation," but closer to published gradients of cell wall matrix deposition. We conclude that the t-SNAREs are transported to the PM on the apical vesicles, but their tip-high gradient alone is insufficient to explain the vesicle fusion gradient in growing tips.     

SNARE complex-mediated degranulation in mast cells

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.