α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin
Figure 3
Preincubation with the cytosolic domain of syntaxin1 abolishes the inhibitory effect of α-SNAP on the AR.
A, SLO-permeabilized human sperm were treated for 15 min at 37°C with increasing concentrations of α-SNAP wild type (closed squares), α-SNAP-(160–295) (open squares), α-SNAP-L294A (open circles), or α-SNAP-M105I (closed circles). Acrosomal exocytosis was initiated by adding 0.5 mM CaCl2 and incubating for a further 15 min. Exocytosis was evaluated by FITC-PSA binding and data normalized (mean ± S.E.M. of at least three independent experiments) as described under “Materials and Methods.” B, SLO-permeabilized sperm were treated for 15 min at 37°C with increasing concentrations of the cytosolic domain of syntaxin1 (residues 1–262). The AR was subsequently initiated by adding 0.5 mM CaCl2 and incubating for 15 min at 37°C. Sperm were stained and the AR scored as in A. Shown is the mean ± S.E.M. of at least three independent experiments. C, SLO-permeabilized sperm were treated for 15 min at 37°C with 300 nM wild type α-SNAP, 50 nM α-SNAP-M105I or 400 nM α-SNAP-L294A pre-mixed with 100 nM syntaxin1's cytosolic domain (black bars). AR was initiated by adding 0.5 mM CaCl2 and incubating for 15 min at 37°C. Several controls were included (gray bars): background AR in the absence of any stimulation (control); AR stimulated by 0.5 mM CaCl2 (calcium); AR inhibited by α-SNAPs (α-SNAPs → calcium); AR unperturbed by a low concentration (100 nM) of syntaxin1 (cyto syntaxin1 → calcium). 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”. Actual percentages of reacted sperm for control and calcium ranged between 11–13 and 23–26% respectively.