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doi: 10.1242/10.1242/jcs.00374

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Regulated secretion: SNARE density, vesicle fusion and calcium dependence

¹ Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
² Unit on Biologic Computation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
³ Department of Physiology and Biophysics, Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

View larger version (41K): [in a new window]   Fig. 1. Full-length western blot probed for syntaxin and SNAP-25. 4 hour control, clostripain- and papain-treated samples (5, 15 and 20 µl load) were evaluated. The normalized fluorescence scans represent the load-adjusted intensity as a function of the normalized western blot coordinate (see Coorssen et al., 2002).

View larger version (24K): [in a new window]   Fig. 2. Effects of exogenous proteases on Ca2+-triggered fusion and CV membrane proteins. (A) Isolated free-floating CVs in suspension were treated with the indicated protease concentrations. Following protease neutralization and washing, recovered CVs were suspended in B-IM buffer. A fraction of each sample was taken for protein extraction and analysis, and the remainder was assayed for Ca2+-triggered fusion. Control CVs (black circle) were sampled and tested immediately after preparation (0 hours; n=16); incubated controls (open circle) were left for 1 hour at 25°C in the absence of protease (n=26). CVs were incubated with 700 (purple triangle; n=5), 3500 (inverted purple triangle; n=9) and 35,000 (purple diamond; n=4) units/ml trypsin, respectively, with 2000 units/ml of papain (blue hexagon; n=9), or with 100 units/ml clostripain (yellow square; n=3) for 1 hour at 25°C. Data presented are means±s.e.m. of the normalized data from a total of 32 separate CV preparations made over the course of two seasons. (B) Isolated CVs, treated as in (A), but with a 4 hour incubation time; control CVs (0 hours) are represented by a solid black curve as in (A). Control samples without protease (x; n=5) were incubated in parallel with samples containing 2000 units/ml papain (blue hexagon; n=6) or 100 units/ml clostripain (yellow square; n=3). (C) Differential effects of exogenous proteases on the profile of hydrophobic proteins extracted from CVs. CVs treated as described in (B) were analyzed by SDS–PAGE and silver staining. Incubations (4 hour) were with buffer only (lane 1), 100 units/ml clostripain (lane 2) or 2000 units/ml papain, as indicated. 1 hour incubations yielded similar protein profiles, although changes were less pronounced (not shown). (D) Endogenously docked CV–PM preparations were treated (1 hour) with the indicated protease concentrations, neutralized, washed, suspended in B-IM buffer and assayed for Ca2+-triggered fusion. Controls (black circle) were sampled and tested 1 hour after preparation, and other samples were incubated (1 hour, 25°C) with 700 (purple triangle), 14,000 (inverted purple triangle) and 35,000 (purple diamond; n=4) units/ml trypsin, respectively, with 3000 units/ml of papain (blue hexagon), or with 100 units/ml clostripain (yellow square). Data are representative examples of one to three determinations.

View larger version (20K): [in a new window]   Fig. 3. Average effects of exogenous proteases on fusion and CV SNARE proteins. Isolated CVs were treated with proteases and their hydrophobic proteins extracted, separated by SDS–PAGE and the constituent SNARE proteins analyzed by quantitative immunoblotting. An aliquot of each sample was also analyzed for fusion. The SNARE complements of isolated CVs are shown in the bar graph following 1-4 hour incubations at 25°C with 2000 units/ml papain (n=10-14 for the individual SNAREs), 100 units/ml clostripain (n=8), or 700 and 3500 units/ml (n=5 each) trypsin, as indicated. Papain treatment reduced VAMP, SNAP-25 and syntaxin to, on average, 341±108, 62±21 and 4±1 copies/CV, respectively. Clostripain reduced VAMP, SNAP-25 and syntaxin to 424±143, 93±17 and 20±5, respectively. Also shown is the extent of post-treatment CV–CV fusion elicited by a maximal [Ca2+]free (1 mM).

View larger version (21K): [in a new window]   Fig. 4. Poisson analysis. Proteolytic loss of SNAREs with increasing trypsin concentrations (1 hour) does not correlate with either the observed decrease in fusion or the derived number of active fusion complexes () at 1 mM [Ca2+]free. Protein proteolysis data are from five separate experiments; the fusion curve and data are from 26 experiments. Proteolytic loss of SNAREs with increasing papain concentrations (1 hour) does not correlate with a loss in fusion. Protein proteolysis and fusion data are from three experiments, except for 2000 units/ml papain, which are the averaged data of 10-14 experiments.

View larger version (34K): [in a new window]   Fig. 5. Proteolytic ablation of syntaxin: average density <1 copy/CV. Using a calibration curve derived from serial dilution of purified glutathione S-transferase (GST)–syntaxin (Coorssen et al., 2001) 0.73 fmoles, corresponding to 113 molecules/CV, were detected in 0 hour controls. No syntaxin was detected in papain-treated (2000 units/ml, replaced every hour for 3 hours) samples. The limit of detection for this calibration was <0.039 fmoles or 0.3 Treated Equivalent (T.E.) molecules/CV for the same protein load (CV number) used to visualize the papain-treated sample. The maximal syntaxin density in this sample is 0.3 molecules/CV. The plot of normalized fluorescence vs. Western Blot Coordinate shows how the signal associated with 0.039 fmoles is easily quantified. The curves are drawn with an offset for visual comparison.

View larger version (16K): [in a new window]   Fig. 6. Ca2+ activity and SNARE density. The midpoint of the Ca2+ activity curve following papain treatment at either 1 hour (blue) or 4 hours (red) is correlated with SNARE density (Copy #/CV). The data were fit using the relationship Axexp(–density/B)+C, where A+C (µM) is the midpoint of the Ca2+ activity curve in the absence of SNAREs (248±10, 433±66 and 305±22) and B is the density decay constant (212±63, 42±10 and 11±3) for VAMP, SNAP-25 and syntaxin, respectively. When SNARE density was normalized by the density decay constant, the pooled transformed data were described by the relationship Axexp(–normalized density/B)+7.1; the midpoint of the Ca2+ activity curve in the absence of SNAREs (A+7.1) is 260±22 µM.

View larger version (15K): [in a new window]   Fig. 7. Kinetics of Ca2+-triggered CV–CV fusion. (A) Differential effects on Ca2+-triggered CV–CV fusion kinetics following 4 hour protease treatments. Papain, but not clostripain, inhibits response to 150 µM [Ca2+]free. However, at 1 mM [Ca2+]free, 100% fusion is observed in all cases. The decrease in the rate and extent of fusion at 150 µM [Ca2+]free is consistent with a reduction in , as determined from a rightward shift in the Ca2+ activity curve. (B) Limiting papain treatment to 3 h alters the rate of fusion elicited with 150 µM [Ca2+]free, with minimal change in the extent of fusion. Thus, the rate of fusion decreased without a substantial change in ; limited papain treatment decreased the fusion probability, p.

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