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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01077

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Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF

Tim Whalley^1,2,*, Kim Timmers^1,*, Jens Coorssen³, Ludmila Bezrukov¹, David H. Kingsley⁴ and Joshua Zimmerberg^1,

¹ Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² Centre for Extracellular Matrix Biology, Department of Biological Sciences, University of Stirling, Stirling, FK9 4LA, UK
³ Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
⁴ United States Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, W.W. Baker Center, Delaware State University, Dover, DE 19901, USA

View larger version (18K): [in a new window]   Fig. 1. Non-hydrolyzable analogues of ATP have no effect on CV exocytosis in vitro. CSCs were prepared in IM and were incubated in the absence of nucleotides (open circles) or with either ATP (closed circles), the poorly hydrolyzable analogue ATP--S (open triangles) or the non-hydrolyzable analogue AppNHp (closed triangles) for 90 minutes at room temperature (20°C) with gentle mixing. Ca 2+-dependent exocytosis was then measured in the continued presence of the added nucleotides. Mean±s.e.m. are shown, n=3.

View larger version (18K): [in a new window]   Fig. 2. Soluble proteins do not reverse inhibition of CV exocytosis by thiol reagents. (A) CSCs were prepared in DTT-free IM and were treated for 1 hour at room temperature (20°C) with gentle mixing with the following. No thiol reagent (open circles), 1 mM NEM (closed circles), 200 µM D10PDP (open triangles) or 50 µM AMSDS (closed triangles). The CSC were centrifuged at 700 g for 1 minute and suspended in fresh IM before the addition of Ca2+ and measurement of exocytosis. Mean±standard error of the mean are shown, n=3 except for AMSDS where mean±standard deviation are shown, n=2. (B) After the treatment described in A, the CSC were washed by a further centrifugation step and were incubated with cytosol at a protein concentration of 10 mg/ml (0.1 µg NSF/mg protein) and 5 mM ATP for 30 minutes. Ca2+ was added and exocytosis measured. Mean±s.e.m. are shown, n=3 except for AMSDS where mean±s.d. are shown, n=2.

View larger version (72K): [in a new window]   Fig. 3. Alignment of amino acid sequences of sea urchin NSF and Chinese hamster NSF. GenBank accession numbers: sea urchin NSF AF171096; Chinese hamster, X15652. An asterisk (*) underneath the sequences indicates amino acid identity, two dots (:) a conservative substitution and a single dot (.) a semi-conservative substitution. Residues in bold are putative nucleotide-binding motifs within each domain (Walker et al., 1982) at residues 264-271 and 547-554 of the sea urchin sequence. The underlined peptide sequence, which spans the D1-D2 domain boundary at residue 490 (Whiteheart et al., 1994), was used to generate polyclonal antibodies.

View larger version (23K): [in a new window]   Fig. 4. Antibody recognizes sea urchin egg cytosol NSF and is inhibited by the immunizing peptide. (A) Affinity-purified anti-NSF antibody was tested against sea urchin egg cytosol by western blotting. 5, 10 or 15 µg of cytosol was subjected to electrophoresis as indicated. Following transfer onto PVDF, the blot was incubated with anti-NSF at a concentration of 1:2000 (0.5 µg/ml) overnight at 4°C. Bound antibody was visualized using alkaline phosphatase-linked second antibody, enhanced chemifluorescence and Storm system fluorescence imaging. The position of putative NSF is indicated with the arrow. (B) 10 µg of cytosol was subjected to electrophoresis and western blot analysis as above but in the presence and absence of the immunizing peptide at a ratio of 3:1 (peptide:IgG). The NSF antibody was incubated on ice for 1 hour with the immunizing peptide prior to incubation with the blot. The labeled arrow indicates the position of the NSF band.

View larger version (21K): [in a new window]   Fig. 5. Cytosolic NSF binds to egg membranes and is inhibited by NSF antibodies. (A) CVs were incubated with cytosol, and with and without (±) nucleotides for 1 hour at room temperature followed by washing in IM ± appropriate nucleotides. Lanes 1, 2, CVs alone; lanes 3, 4, CVs incubated with cytosol without added nucleotide; lanes 5, 6, CVs incubated with cytosol supplemented with 200 µM ATP--S; lanes 7, 8, CVs incubated with cytosol supplemented with 2 mM ATP. (B) IPCs were incubated at room temperature with cytosol at a concentration of 2 mg/ml supplemented with 200 µM ATP--S. NSF antibody was included at concentrations from 0 to 40 µg/ml as indicated. After 1 hour, dishes were extensively rinsed with IM plus 200 µM ATP--S and IPCs dissolved in sample buffer. NSF binding was visualized by western blotting.

View larger version (14K): [in a new window]   Fig. 6. Anti-NSF antibodies do not affect membrane fusion. (A) CSCs were incubated with no antibody (open circles), 50 µg/ml anti-NSF (closed circles) or 50 µg/ml anti-hyalin (open triangles). After 1 hour incubation at room temperature, Ca2+ sensitivity of exocytosis was determined. Mean±standard error of the mean are shown, n=3. (B) CVs were incubated with no antibody (open circles), 50 µg/ml anti-NSF (closed circles) or 50 µg/ml anti-hyalin (open triangles). After 1 hour incubation at room temperature Ca2+ sensitivity of CV/CV fusion was determined. Mean±s.e.m. are shown, n=3.

View larger version (23K): [in a new window]   Fig. 7. CV fusion and NSF binding are differentially sensitive to NEM. Exocytosis in isolated cortices was determined following incubation with various concentrations of NEM for 1 hour at room temperature. Sensitivity of exocytosis to 100 µM Ca2+ was then assessed (open bars). Mean±s.e.m. are shown, n=3. The binding of NSF was determined by incubating cortices with cytosol and 100 µM ATP--S. Prior to the binding assay, cytosol was treated for 1 hour at room temperature with the same concentrations of NEM used to inhibit CV exocytosis. NSF binding was determined by western blotting with anti-NSF antibody followed by densitometric scanning (filled bars).

View larger version (44K): [in a new window]   Fig. 8. NSF is present in isolated planar cortex but is not associated with CVs. (A) Replicate western blots probed for sea urchin NSF, calreticulin and two CV cargo proteins. Lane 1, IPC containing 1.5x107 CVs; lane 2, an equivalent amount of cortex denuded of CVs; lanes 3-5, 1.5x107 isolated CVs (three different preparations); lane 6, egg cytosol (5 µg total protein); lane 7, egg microsomes (10 µg total protein). (B) NSF western blot. Lane 1, egg cytosol (5 µg total protein); lanes 2-6, recombinant NSF-His6 standard curve (0.48, 0.24, 0.12, 0.048, and 0.024 fmol as hexamer); lanes 7, 8, two preparations of 1.5x107 isolated CVs. (C) Recovery in western blots of recombinant sea urchin NSF added to cortical vesicle material. Lane 1, 0.024 fmol NSF alone; lane 2, 0.048 fmol NSF; lane 3, CV proteins; lane 4, CV proteins plus 0.024 fmol NSF; lane 5, CV proteins plus 0.048 fmol NSF. Representative of two experiments.