α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin
Figure 8
α-SNAP prevents docking of the outer acrosomal to the plasma membrane.
To investigate the effect of α-SNAP on the close appositions between the outer acrosomal and plasma membranes required for exocytosis, SLO-permeabilized human sperm were treated for 15 min at 37°C with 500 nM wild type α-SNAP before challenging with 0.5 mM CaCl2 (α-SNAP → calcium). We designed the following controls in an effort to validate each aspect of the experiments: i) untreated cells as control for the morphology of unswollen, undocked acrosomes (not shown); ii) 200 nM complexin II for 15 min at 37°C followed by 0.5 mM CaCl2 as a control for the morphology of swollen, docked acrosomes (only visible in unreacted cells, hence the choice of late acting AR blockers, “complexin → calcium”); and iii) 500 nM α-SNAP-(160–295) plus 20 µM BAPTA-AM followed by 0.5 mM CaCl2 (BAPTA-AM is necessary to prevent acrosomal loss due to exocytosis because the truncated version of α-SNAP is not an AR blocker, “BAPTA-AM → α-SNAP-(160–295) → calcium”). The distance between the outer acrosomal and plasma membranes was measured at the edge of acrosomal invaginations only in images where the membrane bilayers were clearly distinguished. We analyzed 10 cells, 34 contacts, 2 experiments for the untreated control; 16 cells, 48 contacts, 2 experiments, for complexin; 31 cells, 99 contacts, 3 experiments for α-SNAP; and 19 cells, 63 contacts, 2 experiments for α-SNAP-(160–295). Distance distributions are plotted as histograms below each electron micrograph and compared using the Kolmogorov-Smirnov test for two sets of data. Three different distribution patterns were identified: one corresponding to the distance distribution in untreated cells (control); one in cells treated with complexin or BAPTA-AM plus α-SNAP-(160–295); and one in cells treated with wild type α-SNAP (p<0.01 compared with the histogram of cells treated with complexin). pm = plasma membrane; oam = outer acrosomal membrane. Scale bars = 100 nm.