Figure 1.
α-SNAP halts human sperm AR before SNAREs assemble in trans complexes.
A, SLO-permeabilized sperm were treated for 15 min at 37°C with 300 nM α-SNAP before initiating acrosomal exocytosis with 300 nM GTP-γ-S-bound Rab3A and incubating at 37°C for 15 min (black bar). Controls (gray bars) included: background AR in the absence of any stimulation (control); AR stimulated by 0.5 mM CaCl2 (calcium) or 300 nM GTP-γ-S-bound Rab3A (Rab3A); and AR inhibited by 300 nM α-SNAP (α-SNAP → calcium). Sperm were fixed and stained with FITC-PSA. Exocytosis was evaluated by FITC-PSA binding and data normalized (mean ± S.E.M, three independent experiments) as described under “Materials and Methods.” Actual percentages of reacted sperm for control and calcium ranged between 11–16 and 20–26% respectively. B, SLO-permeabilized sperm were loaded with 10 µM NP-EGTA-AM (NP-EGTA) for 10 min at 37°C to chelate intravesicular calcium. Next, the AR was initiated by adding 0.5 mM CaCl2 and samples incubated for further 10 min at 37°C. This protocol allows exocytosis to proceed up to the intra-acrosomal calcium-sensitive step, which for SNARE proteins means disassembly of pre-existing cis complexes and reassembly of the monomers in loose trans arrays. At this point, sperm were treated for 10 min with 300 nM α-SNAP. All these procedures were carried out in the dark. UV 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 (black bar). 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 300 nM α-SNAP (α-SNAP → calcium); inhibitory effect of NP-EGTA-AM in the dark (NP-EGTA → calcium → dark) and recovery upon illumination (NP-EGTA → calcium → hν); and the inhibitory effect of α-SNAP when present throughout the experiment (NP-EGTA → α-SNAP → calcium → hν). Sperm were stained and the AR scored as described in A. Actual percentages of reacted sperm for control and calcium ranged between 13–17 and 25–26% respectively. Shown is the mean ± S.E.M. of at least three independent experiments.
Figure 2.
α-SNAP-M105I binds syntaxin with higher affinity than wild type α-SNAP.
A, 4 µg recombinant α-SNAP (wild type and mutants) were immobilized by binding to the surface of polypropylene microcentrifuge tubes for 20 min at 20°C. Excess protein was removed and non-specific binding sites were blocked as described under “Materials and Methods” before incubating with 2 µg recombinant NSF for 10 min at 4°C. All tubes were then washed and the immobilized proteins were recovered by boiling for 5 min in Laemmli sample buffer. Proteins were resolved on 10% SDS-polyacrylamide gels and analyzed by anti-NSF (top) and anti-α/β-SNAP (loading control, bottom) Western blot. On the far right lane we ran recombinant NSF as a control. Shown is an experiment representative of three repetitions. B, left, syntaxin1 (0.9 µM) was incubated with 5 µM wild type α-SNAP, 5 µM α-SNAP-(160–295), 5 µM α-SNAP-L294A, or 2.5 µM α-SNAP-M105I in a buffer containing 5 mM MgCl2 for 2 h prior to addition of 0.3 µM NSF together with 2 mM ATP or ATP-γ-S. After an additional 1 h at 30°C, syntaxin was collected by inmunoprecipitation as described under “Materials and Methods.” Precipitated protein complexes were separated on 10% SDS-polyacrylamide gels and immunoblotted with the anti-α/β-SNAP (top) or the monoclonal anti-syntaxin1 (bottom) antibodies. LC, immunoglobulin light chain, HC, immunoglobulin heavy chain,* indicates the electrophoretic mobility of α-SNAP-(160–295). Mr standards (×103) are indicated on the left. Shown is an experiment representative of three repetitions. Right, densitometric analysis of Western blots for α-SNAP (mean ± SEM, n = 3) showing the fraction of α-SNAP coimmunoprecipitated with syntaxin normalized to the amount of syntaxin in each sample. Gray bars, control amount of syntaxin-bound α-SNAP when ATP hydrolysis was prevented (ATP-γ-S lanes, set to 100% for each protein version); black bars, syntaxin-bound α-SNAP after NSF/ATP-driven disassembly expressed as a percentage of the amount precipitated when ATP hydrolysis was prevented. ** p<0.01 for α-SNAP wt ATP vs ATP-γ-S and * p<0.05 for α-SNAP-M105I ATP vs ATP-γ-S (Student's t-test for single group mean); p<0.01 for α-SNAP-M105I ATP vs α-SNAP wt ATP (Student's t-test for unpaired comparison). C, Syntaxin1 was incubated with the indicated concentrations of wild type α-SNAP or M105I as in B, except that NSF and ATP were omitted, and samples were processed for syntaxin immunoprecipitation and Western blot. Shown is a blot (out of four repetitions) probed for α-SNAP (top) and syntaxin (bottom). Right, densitometric analysis of Western blots including that depicted in C (mean ±S.E.M., n = 4) showing the maximal amount of α-SNAP coimmunoprecipitated with syntaxin as a function of the initial concentration added to each reaction mixture. Each value was normalized taking into account syntaxin's densitometric signal in the corresponding lane.
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.
Figure 4.
NSF rescues the AR block imposed by the M105I mutant at high NSF/α-SNAP ratios.
A, SLO-permeabilized sperm were loaded for 15 min at 37°C with 300 nM wild type α-SNAP or 400 nM α-SNAP-L294A followed by 300 nM NSF and the reaction mixtures were incubated at 37°C for 15 min. The AR was initiated by adding 0.5 mM CaCl2 and incubating for 15 min at 37°C (black bars). Controls (gray bars) included: background AR in the absence of any stimulation (control); AR stimulated by 0.5 mM CaCl2 (calcium); AR inhibited by 300 nM wild type α-SNAP or 400 nM α-SNAP-L294 (α-SNAP wt/L294A → calcium); and AR unperturbed by 300 nM NSF (NSF → 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.” B, SLO-permeabilized spermatozoa were loaded with the indicated concentrations of α-SNAP-M105I for 15 min at 37°C and subsequently challenged with 0.5 mM CaCl2 for 10 min at 37°C. NSF was then added as indicated in the labels and incubations continued for an additional 10 min (black bars). 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-SNAP-M105I (α-SNAP-M105I 50/100 nM → calcium); and AR unperturbed by NSF (NSF 300/500 nM → calcium). Sperm were stained and the AR scored as in A. Shown is the mean ± S.E.M. of three independent experiments. Actual percentages of reacted sperm for control and calcium ranged between 12–16 and 26–28% respectively.
Figure 5.
α-SNAP does not interfere with sperm SNARE complex disassembly.
Permeabilized spermatozoa were loaded for 15 min at 37°C with 300 nM α-SNAP wild type (M, N, O), 50 nM α-SNAP-M105I (S, T, U), or 400 nM α-SNAP-L294A (V, W, X), subsequently treated with 100 nM BoNT/C and finally activated with 0.5 mM CaCl2. The cells were then fixed and triple stained with the rabbit polyclonal anti-syntaxin1A antibody (that recognizes an epitope located in a portion of the molecule released by the toxin; “anti-syx 1A”, red, left panels), FITC-PSA (to differentiate between reacted and intact sperm; green, central panels), and Hoechst 33342 (to visualize all cells in the field; blue, right panels). Notice that reacted sperm were negative for syntaxin1A staining (arrowheads in panels P and V). Asterisks in panels G, M and S indicate cells with intact acrosomes but without syntaxin1 immunostaining due to toxin cleavage. BoNT/C had no effect on syntaxin1A staining in resting sperm (A). Labeling in sperm stimulated with calcium was significantly reduced by the wild type (G) but not by the catalytically dead (J, P) BoNT/C. Bars = 5 µm. Y, Quantification of the percentage of cells exhibiting syntaxin1 acrosomal staining from three independent experiments (mean ± S.E.M.).
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.
Figure 7.
Recombinant α-SNAP halts the AR at a stage when SNAREs are monomeric.
A, we incubated SLO-permeabilized sperm with 300 nM α-SNAP for 10 min at 37°C followed by 100 nM light chain of TeTx, and 0.5 mM CaCl2 for an additional 10 min (G, H, I). To evaluate whether preincubation with PTP1B ultimately leads to cis SNARE complex disassembly, we loaded sperm with this phosphatase (27 nM) and treated with TeTx without an exocytotic trigger (J, K, L). We then fixed and triple stained the cells with an anti-synaptobrevin2 antibody (“anti-syb 2”, red, left panels), FITC-PSA (to differentiate between reacted and intact sperm; green, central panels), and Hoechst 33342 (to visualize all cells in the field; blue, right panels). Asterisks in D, G and J indicate cells with intact acrosomes but without synaptobrevin2 immunostaining due to toxin cleavage. TeTx did not cleave synaptobrevin in resting sperm (A) but substantially decreased head labeling in sperm stimulated with calcium (D). Bars = 5 µm. M, percentage of cells showing synaptobrevin2 acrosomal staining from three independent experiments (mean ± S.E.M.). N, SLO-permeabilized spermatozoa were loaded with 27 nM PTP1B and incubated for 15 min at 37°C to disassemble cis SNARE complexes. Next, we added 300 nM α-SNAP and incubated for a further 15 min at 37°C. Finally, the AR was initiated by adding 0.5 mM CaCl2 and incubating for an additional 15 min (black bar). Controls (gray bars) include: background AR in the absence of any stimulation (control); AR stimulated by 0.5 mM CaCl2 (calcium); AR inhibited by 300 nM wild type α-SNAP (α-SNAP → calcium); and AR unperturbed by 27 nM PTP1B (PTP1B → calcium). Sperm were fixed and stained with FITC-PSA and the AR scored as described in the legend to Figure 1. Shown is the mean ± range of two independent experiments. O, we incubated SLO-permeabilized sperm with 300 nM wild type (top) or 50 nM M105I (bottom) 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 TeTx 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-SNAPs block with 300 nM NSF for 10 min at 37°C (black bars). To confirm that recombinant α-SNAP does not stimulate endogenous NSF to disassemble sperm cis SNARE complexes before adding an AR trigger (to dephosphorylate NSF), 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) and 50 nM α-SNAP-M105I (α-SNAP-M105I → calcium); AR unperturbed by 300 nM NSF (NSF → calcium); AR inhibited by α-SNAPs and rescued by NSF (α-SNAPs → calcium → NSF); and AR inhibited by 100 nM TeTx (TeTx → calcium). Sperm were stained and the AR scored as described in the legend to Figure 1. Shown is the mean ± S.E.M. of at least three independent experiments. Actual percentages of reacted sperm for control and calcium ranged between 10–18 and 21–26% respectively.
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.