α-Synuclein is required for sperm exocytosis at a post-fusion stage.

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

A connection between reversible tyrosine phosphorylation and SNARE complex disassembly activity of N-ethylmaleimide-sensitive factor unveiled by the phosphomimetic mutant N-ethylmaleimide-sensitive factor-Y83E.

N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor (SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF is constitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF-Y83E inhibits secretion in the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our model is human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosine phosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitation increased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomal domain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF-Y83E bound PTP1B and thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complex disassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF-Y83E prevents endogenous PTP1B from dephosphorylating sperm NSF, thus maintaining NSF's activity in a repressed mode and the SNARE complex unable to dissociate. The contribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosine phosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generated direct evidence that explains the dominant-negative role of the phosphomimetic mutant NSF-Y83E.

Grab recruitment by Rab27A-Rabphilin3a triggers Rab3A activation in human sperm exocytosis.

Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.

The proteins of exocytosis: lessons from the sperm model.

Exocytosis is a highly regulated process that consists of multiple functionally, kinetically and/or morphologically definable stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13 and -18, complexins and synaptotagmins. In the present article, the molecules of exocytosis are reviewed, using human sperm as a model system. Sperm exocytosis is driven by isoforms of the same proteinaceous fusion machinery mentioned above, with their functions orchestrated in a hierarchically organized and unidirectional signalling cascade. In addition to the universal exocytosis regulator calcium, this cascade includes other second messengers such as diacylglycerol, inositol 1,4,5-trisphosphate and cAMP, as well as the enzymes that synthesize them and their target proteins. Of special interest is the cAMP-binding protein Epac (exchange protein directly activated by cAMP) due in part to its enzymatic activity towards Rap. The activation of Epac and Rap leads to a highly localized calcium signal which, together with assembly of the SNARE complex, governs the final stages of exocytosis. The source of this releasable calcium is the secretory granule itself.

Molecular mechanisms of membrane fusion during acrosomal exocytosis.

Sperm are attractive cells. Understanding their physiology has motivated researchers from all over the globe for decades. Initially came the description of sperm's overall shape and properties, together with their genesis and development in the testis. Later, the study of exocytosis took off owing to ultrastructural analysis that achieved exquisite levels of detail. Biochemical analysis ensued, identifying ligands and signalling pathways whose end point was exocytosis. Somehow, the unveiling of the molecular mechanisms involved in membrane fusion itself lagged behind all this progress. The picture changed dramatically in the last few years, due to an explosion in our knowledge of the many proteins required for exocytosis and its regulation, and the discovery that very similar versions of these proteins play the same roles in virtually all membrane fusion models. Luckily, sperm are not the exception to this rule. For instance, fusion of the outer acrosomal to the plasma membrane depends on Rab3 activation, alpha-SNAP/NSF, synaptotagmin, and SNAREs; it also requires an efflux of calcium from the acrosomal lumen. Convergence of Rab- and toxin-sensitive SNARE-dependent pathways is a hallmark of the acrosome reaction that makes it an attractive mammalian model to study the different phases of the membrane fusion cascade. Finally, because nature has endowed sperm with a cellular specialization that gives them a single, irreversible chance to fertilise an egg, the acrosome reaction is more straightforward to dissect than fusion in other cell types, where the same substances are secreted again and again, requiring the membranes and fusion machinery to recycle multiple times.