SNAREs Are Assembled in Neurotoxin-Resistant Complexes in Resting Spermatozoa (original) (raw)

2013

Abstract

<div><p>(A) Permeabilized spermatozoa were treated at 37 °C for 15 min with 357 nM BoNT/E, 100 nM BoNT/B, or 100 nM TeTx. Next, 2.5 μM TPEN (see [B]) was added and AE was activated by adding 0.5 mM CaCl₂ (10 μM free Ca²⁺) and the incubation continued for an additional 15 min (black bars). 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>. Several controls were included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺); TPEN effect on exocytosis (TPEN→Ca²⁺); inhibitory effect of the neurotoxins on exocytosis (neurotoxin→Ca²⁺); and block of neurotoxin activity by TPEN (TPEN→neurotoxin→Ca²⁺).</p> <p>(B) Recombinant SNAP25 (0.7 μg) was incubated for 15 min at 37 °C in the presence of 0.6 μg of BoNT/E and increasing concentrations of TPEN. Samples were then resolved by SDS-PAGE and stained with Coomassie blue. Molecular weight standards are indicated on the left (in kilodaltons). Densitometry and quantitation of the stained bands show 100%, 7%, 92%, 98%, 100%, and 100% of intact SNAP25 in lanes 1–6 (from left to right), respectively.</p> <p>(C) Treatment with TeTx was performed as described in (A), in the presence of 310 nM NSF and 500 nM α-SNAP (NSF/αS) to promote SNARE complex dissociation (black bar). Incubation with NSF/α-SNAP in the presence of TPEN-inactivated toxin did not affect exocytosis (grey bar).</p> <p>The data in (A) and (C) 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#st003" target="_blank">Table S3</a>.</p></div

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