α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin
Figure 6
Recombinant α-SNAP halts the AR downstream of SNARE complex disassembly.
A, to investigate whether α-SNAP impairs cis SNARE complex disassembly (which renders syntaxin sensitive to BoNT/C cleavage), we incubated SLO-permeabilized sperm with 300 nM wild type α-SNAP for 10 min at 37°C followed by 0.5 mM CaCl2 for an additional 10 min. Subsequently, we added 100 nM light chain of BoNT/C (wild type, top, or catalytically dead mutant, bottom) and incubated for a further 10 min; we stopped toxin activity with 2.5 µM TPEN for 10 min. At the end of the incubation we released the α-SNAP block with 300 nM NSF for 10 min at 37°C (black bars). To address whether recombinant α-SNAP stimulates endogenous NSF to disassemble sperm cis SNARE complexesm even before adding an AR trigger (which leads to NSF's dephosphorylation), we modified the order of addition of reagents as indicated in the figure's labels and incubated as described above (open bars). We included the following controls (gray bars): background AR in the absence of any stimulation (control); AR stimulated by 0.5 mM CaCl2 (calcium); AR inhibited by 300 nM α-SNAP (α-SNAP → calcium); AR unperturbed by 300 nM NSF (NSF → calcium); AR inhibited by α-SNAP and rescued by NSF (α-SNAP → calcium → NSF); and AR inhibited by 100 nM wild type (BoNT/C → calcium) but not by the inactive (BoNT/C-E230A → calcium) neurotoxins. Cells were fixed, acrosomal exocytosis was evaluated by FITC-PSA binding and data were normalized (mean ± S.E.M. of at least three independent experiments) as described under “Materials and Methods.” B, we conducted experiments identical to those depicted in panel A, except that we applied 50 nM α-SNAP-M105I instead of 300 nM wild type α-SNAP. We included the following controls (gray bars): AR inhibited by 50 nM α-SNAP-M105I (α-SNAP-M105I → calcium); AR unperturbed by 300 nM NSF (NSF → calcium); AR inhibited by α-SNAP-M105I and rescued by NSF (α-SNAP-M105I → calcium → NSF); and AR inhibited by 100 nM wild type (BoNT/C → calcium) neurotoxin. Sperm were stained and the AR scored as described in A. Shown is the mean ± S.E.M. of at least three independent experiments. Actual percentages of reacted sperm for control and calcium ranged between 7–11 and 19–21% respectively.