FIGURE 5.

Recombinant Munc18-1 halts acrosomal exocytosis at a stage when SNAREs are monomeric. A and B, SLO-permeabilized spermatozoa were loaded with 27 nm PTP1B (A) or 300 nm NSF (B) and incubated for 15 min at 37 °C to disassemble cis-SNARE complexes. Next, we added 4 nm Munc18-1 and incubated for a further 15 min at 37 °C. Finally, we added 0.5 mm CaCl₂ and incubated for an additional 15 min to stimulate exocytosis (black bars). Controls (gray bars) included the following: background exocytosis in the absence of any stimulation (control); exocytosis stimulated by 0.5 mm CaCl₂ (calcium); exocytosis inhibited by 4 nm Munc18-1 and unperturbed by 27 nm PTP1B or 300 nm NSF. Sperm were fixed, and acrosomal exocytosis was evaluated using PSA-FITC, and data normalized as described under “Experimental Procedures.” The values represent mean ± S.E. of at least three independent experiments. C–G, we incubated SLO-permeabilized sperm under resting conditions (C and D) or with 300 nm NSF to promote cis-SNARE complex disassembly (E). At the end of the incubation, we added 100 nm light chain of TeTx to test VAMP2/syb2 neurotoxin sensitivity (D and E). Other aliquots were incubated with 10 μm BAPTA-AM (B-AM, F and G). When indicated, 4 nm Munc18-1 was added to the assay (G). Next, we added 0.5 mm CaCl₂ to stimulate exocytosis. At the end of the incubation, we added 100 nm light chain of TeTx. We then fixed and triple-stained the cells with an anti-VAMP2/syb2 antibody (red, left panels), PSA-FITC (to differentiate between reacted and intact sperm; green, central panels), and Hoechst 33342 (to visualize all cells in the field; blue, right panels). Asterisks indicate cells with intact acrosomes but without VAMP2/syb2 immunostaining due to toxin cleavage. Bars, 5 μm. H, quantification of the percentage of cells treated as in C–G exhibiting VAMP2/syb2 acrosomal staining from three independent experiments (mean ± S.E.).