Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal Exocytosis

The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca(2+) chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca(2+) entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca(2+) must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis

α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin

α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs — wild type and M105I — inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160–295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is essential for docking

Sperm from Hyh Mice Carrying a Point Mutation in αSNAP Have a Defect in Acrosome Reaction

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of αSNAP has been detected in these animals. αSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had αSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type αSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant αSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of αSNAP, and that a fully functional αSNAP is necessary for acrosomal exocytosis, a key event in fertilization

SNAREs Are Assembled in Neurotoxin-Resistant Complexes in Resting Spermatozoa

<div><p>(A) Permeabilized spermatozoa were treated at 37 °C for 15 min with 357 nM BoNT/E, 100 nM BoNT/B, or 100 nM TeTx. Next, 2.5 μM TPEN (see [B]) was added and AE was activated by adding 0.5 mM CaCl₂ (10 μM free Ca²⁺) and the incubation continued for an additional 15 min (black bars). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. Several controls were included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺); TPEN effect on exocytosis (TPEN→Ca²⁺); inhibitory effect of the neurotoxins on exocytosis (neurotoxin→Ca²⁺); and block of neurotoxin activity by TPEN (TPEN→neurotoxin→Ca²⁺).</p> <p>(B) Recombinant SNAP25 (0.7 μg) was incubated for 15 min at 37 °C in the presence of 0.6 μg of BoNT/E and increasing concentrations of TPEN. Samples were then resolved by SDS-PAGE and stained with Coomassie blue. Molecular weight standards are indicated on the left (in kilodaltons). Densitometry and quantitation of the stained bands show 100%, 7%, 92%, 98%, 100%, and 100% of intact SNAP25 in lanes 1–6 (from left to right), respectively.</p> <p>(C) Treatment with TeTx was performed as described in (A), in the presence of 310 nM NSF and 500 nM α-SNAP (NSF/αS) to promote SNARE complex dissociation (black bar). Incubation with NSF/α-SNAP in the presence of TPEN-inactivated toxin did not affect exocytosis (grey bar).</p> <p>The data in (A) and (C) were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st003" target="_blank">Table S3</a>.</p></div

VAMP2 Is Engaged in Loose SNARE Complexes before the Efflux of Intra-Acrosomal Ca²⁺

<p>Permeabilized spermatozoa were loaded with 10 μM BAPTA-AM (B-AM) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺). After 15 min incubation at 37 °C to allow exocytosis to proceed to the intra-acrosomal Ca²⁺-sensitive step, 100 nM neurotoxins recognizing VAMP (BoNT/B or TeTx) were added to the tubes and the samples were incubated for 15 min at 37 °C (B-AM→Ca²⁺→neurotoxin, black bars). Samples were then immunolabeled with an anti-VAMP2 antibody as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. Notice that at this stage VAMP2 immunolabeling was sensitive to BoNT/B but not to TeTx. Several other conditions are included (grey bars). The toxins did not affect VAMP2 staining in resting sperm (compare control versus B-AM→neurotoxin). However, the toxins decreased the VAMP2 labeling when present during stimulation (B-AM→neurotoxin→Ca²⁺). Fluorescence was normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st008" target="_blank">Table S8</a>.</p

SNAREs Reassemble in Loose Complexes That Are Resistant to NSF/α-SNAP before the Efflux of Intra-Acrosomal Ca²⁺

<div><p>(A) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺). After 15 min incubation at 37 °C to allow exocytosis to proceed to the intra-acrosomal Ca²⁺-sensitive step, 800 nM recombinant SNAP25 (SNAP25) was added to compete with endogenous SNAP25. Intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺→SNAP25→hν, black bar). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A). After 15 min incubation at 37 °C, 100 nM neurotoxin recognizing VAMP (BoNT/B and TeTx) was added to the tubes to assess whether the SNAREs had reassembled in loose <i>trans</i> complexes sensitive to BoNT/B but not to TeTx. After 15 min incubation at 37 °C, intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺/Rab3A→neurotoxin→hν, black bars). Sperm were then fixed and AE measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(C) To assess whether NSF/α-SNAP can disassemble loose <i>trans</i> SNARE complexes, permeabilized sperm treated as in (B) were incubated with TeTx in the presence of 310 nM NSF and 500 nM α-SNAP (NP→Ca²⁺/Rab3A→NSF/αS+TeTx→hν, black bars).</p> <p>Several controls were included in (A), (B), and (C) (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A); inhibitory effect of NP-EGTA-AM in the dark (NP→Ca²⁺/Rab3A→dark) and the recovery upon illumination (NP→Ca²⁺/Rab3A→hν); inhibitory effect when SNAP25 was present throughout the incubations (NP→SNAP25→Ca²⁺→hν); inhibitory effect when the neurotoxins were present throughout the incubations (NP→neurotoxin→Ca²⁺/Rab3A→hν); and the effect of NSF/α-SNAP on SNARE complexes in unstimulated sperm (NSF/αS+TeTx→TPEN→Rab3A→hν). The data were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st007" target="_blank">Table S7</a>.</p></div

Rab3A Is Required before, and SNAREs and Synaptotagmin VI after, Intra-Acrosomal Ca²⁺ Efflux

<p>Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 0.5 mM CaCl₂ (10 μM free Ca²⁺)(Ca²⁺). After further 15 min incubation at 37 °C to allow exocytosis to proceed up to the intra-acrosomal Ca²⁺-sensitive step, sperm were treated for 15 min at 37 °C with antibodies that recognize Rab3A (20 μg/ml, anti-Rab3A), SNAP25 (20 μg/ml, anti-SNAP25), syntaxin1A (1/25 dilution, anti-Stx1A), VAMP2 (20 μg/ml, anti-VAMP2), or synaptotagmin VI (30 μg/ml, anti-StgVI). All these procedures were carried out in the dark. UV flash photolysis of the chelator was induced at the end of the incubation period (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺→antibody→hν, black bars; a diagram of the experiment is shown at the top of the figure). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. Several controls were included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺), inhibitory effect of NP-EGTA-AM in the dark (NP→Ca²⁺→dark), and the recovery upon illumination (NP→Ca²⁺→hν); and inhibitory effect of the antibodies when present throughout the experiment (NP→antibody→Ca²⁺→hν). The data were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st002" target="_blank">Table S2</a>.</p

Effect of BoNT/B and TeTx on VAMP2 Immunofluorescence

<p>Sperm were incubated with 100 nM BoNT/B or TeTx (15 min at 37 °C) as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#pbio-0030323-g008" target="_blank">Figure 8</a>. The cells were then triple-stained with an anti-VAMP2 antibody that recognizes an epitope that is cleaved by the toxin (red; [A, D, G, J, M, P, and S]), FITC-PSA to differentiate between reacted and intact sperm (green; [B, E, H, K, N, Q, and T]), and Hoechst 33258 to visualize all cells in the field (blue; [C, F, I, L, O, R, and U]). BoNT/B and TeTx had no effect on resting sperm (compare [D–F] and [M–O] with [A–C]). However, labeling in sperm stimulated with 10 μM Ca²⁺ in the presence of BAPTA-AM to prevent exocytosis (observe that the PSA staining is not affected) was significantly reduced by the toxins (asterisks, [G] and [P]). In contrast, when cells were first allowed to arrive at the intra-acrosomal Ca²⁺-sensitive step and then treated with toxins, BoNT/B caused a significant decrease of the VAMP2 label (asterisks, [J]), whereas TeTx had no effect (S). Bars = 5 μm.</p

Syntaxin1A Is Assembled in Toxin-Resistant Complexes That Are Disassembled by NSF/α-SNAP or by Sperm Activation

<div><p>(A) Permeabilized spermatozoa were incubated for 15 min at 37 °C with increasing concentrations of BoNT/C (black circles, wild type; grey circles, EA, a protease-inactive mutant) and then stimulated with 10 μM Ca²⁺ for 15 min at 37 °C. Afterwards, sperm were fixed and AE measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) To assess the assembly state of syntaxin1A, sperm were incubated with 100 nM BoNT/C (15 min at 37 °C), and the cells were then fixed and immunostained with an anti-syntaxin1A antibody recognizing an epitope that is cleaved by the toxin. To prevent AE, which would release syntaxin into the medium by vesiculation of the acrosome, intra-acrosomal Ca²⁺ was chelated with 10 μM BAPTA-AM (15 min at 37 °C, B-AM). The toxin treatment in resting sperm (BoNT/C) or B-AM-loaded sperm (B-AM→BoNT/C) had no effect on the syntaxin labeling compared to untreated sperm (control). However, when 310 nM NSF and 500 nM α-SNAP were added to the system to promote the disassembly of SNARE complexes, the toxin significantly decreased the syntaxin labeling (B-AM→NSF/αS→BoNT/C). The BoNT/C treatment also affected syntaxin labeling when sperm were stimulated for 15 min at 37 °C with 10 μM free Ca²⁺ (B-AM→ BoNT/C→Ca²⁺) or 300 nM Rab3A (B-AM→BoNT/C→Rab3A). The protease-inactive mutant did not affect labeling under these conditions (B-AM→BoNT/C-EA→Ca²⁺). Fluorescence was normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. The data represent the mean ± SEM. Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st005" target="_blank">Table S5</a>.</p></div

Effect of BoNT/C on Syntaxin1A Immunofluorescence

<p>Sperm were incubated with 100 nM BoNT/C (15 min at 37 °C) as explained in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#pbio-0030323-g004" target="_blank">Figure 4</a>. The cells were then fixed and triple-stained with an anti-syntaxin1A antibody that recognizes an epitope trimmed by the toxin (red; [A, D, G, and J]), FITC-PSA to differentiate between reacted and intact sperm (green; [B, E, H, and K]), and Hoechst 33258 to visualize all cells in the field (blue; [C, F, I, and L]). Notice that spontaneously reacted sperm were negative for syntaxin1A staining (arrowheads in [D] and [E]). BoNT/C had no effect on resting sperm (compare [A–C] with [D–F]). However, labeling in sperm stimulated with 10 μM Ca²⁺ in the presence of BAPTA-AM to prevent exocytosis (observe that PSA staining is not affected) was significantly reduced by the toxin (asterisks, [G]). In contrast, the same experimental condition in the presence of the protease-inactive toxin (BoNT/C-EA) had no effect (J–L). Bars = 5 μm.</p