SNAREs Reassemble in Loose Complexes That Are Resistant to NSF/α-SNAP before the Efflux of Intra-Acrosomal Ca²⁺ (original) (raw)

2013

Abstract

<div><p>(A) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺). After 15 min incubation at 37 °C to allow exocytosis to proceed to the intra-acrosomal Ca²⁺-sensitive step, 800 nM recombinant SNAP25 (SNAP25) was added to compete with endogenous SNAP25. Intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺→SNAP25→hν, black bar). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A). After 15 min incubation at 37 °C, 100 nM neurotoxin recognizing VAMP (BoNT/B and TeTx) was added to the tubes to assess whether the SNAREs had reassembled in loose <i>trans</i> complexes sensitive to BoNT/B but not to TeTx. After 15 min incubation at 37 °C, intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺/Rab3A→neurotoxin→hν, black bars). Sperm were then fixed and AE measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(C) To assess whether NSF/α-SNAP can disassemble loose <i>trans</i> SNARE complexes, permeabilized sperm treated as in (B) were incubated with TeTx in the presence of 310 nM NSF and 500 nM α-SNAP (NP→Ca²⁺/Rab3A→NSF/αS+TeTx→hν, black bars).</p> <p>Several controls were included in (A), (B), and (C) (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A); inhibitory effect of NP-EGTA-AM in the dark (NP→Ca²⁺/Rab3A→dark) and the recovery upon illumination (NP→Ca²⁺/Rab3A→hν); inhibitory effect when SNAP25 was present throughout the incubations (NP→SNAP25→Ca²⁺→hν); inhibitory effect when the neurotoxins were present throughout the incubations (NP→neurotoxin→Ca²⁺/Rab3A→hν); and the effect of NSF/α-SNAP on SNARE complexes in unstimulated sperm (NSF/αS+TeTx→TPEN→Rab3A→hν). The data were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st007" target="_blank">Table S7</a>.</p></div

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