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Ca²⁺-induced changes in SNAREs and synaptotagmin I correlate with triggered exocytosis from chromaffin cells: insights gleaned into the signal transduction using trypsin and botulinum toxins

Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, UK

View larger version (53K): [in a new window]   Fig. 1. Ca2+ induces changes in the conformation of SNAREs in permeabilised chromaffin cells: acquisition of resistance to trypsin correlates with the extent of exocytosis. Chromaffin cells permeabilised using 20 µM digitonin in KGEP without () or with 2 mM MgATP ([UNK]) and the indicated buffered, free [Ca2+]. After 15 minutes, aliquots were assayed (±s.d, n=4; some error bars are obscured by symbols) for released catecholamine (A). The cells were maintained for a further 30 minutes, 2 mM PMSF was added and a membrane fraction prepared from four wells; after boiling for 2 minutes in 1% SDS, the samples were subjected to SDS-PAGE and western blotting, with antibodies reactive with the specified proteins (B). Only the relevant track positions are shown. (C) Western blots of membranes from cells treated as above, except that 100 µg/ml trypsin was added to the KGEP immediately after the removal of aliquots for the catecholamine assay; thus, the cells were exposed to the protease for 30 minutes. (D,E) The intensity of all the immunosignals, in cells exposed to trypsin in the absence of MgATP, were computed from digitised images (see Materials and Methods). For each protein, the values were normalised as a percentage of the highest intensity signal, then plotted against [Ca2+]; values for DßH are plotted in (D) while synaptotagmin I (), syntaxin (), SNAP-25 () and synaptobrevin () are shown in (E). Plotted data are representative of results obtained on at least three separate occasions.

View larger version (39K): [in a new window]   Fig. 2. SNAREs in cells subjected to run-down do not acquire resistance to trypsin. (A) Cells were permeabilised with digitonin in KGEP containing Ca2+ at the indicated concentrations () or with digitonin in KGEP lacking Ca2+ and maintained for 30 minutes before the addition of the cation ([UNK]). In both cases, aliquots were removed for catecholamine assay 15 minutes after the application of Ca2+ and released catecholamine was calculated (±s.d., n=4; some error bars are obscured by symbols). Trypsin was immediately added (100 µg/ml final concentration) and incubated for 30 minutes before adding 2 mM PMSF, harvesting the cells and isolating a membrane-enriched fraction. The latter samples were boiled for 2 minutes before being subjected to SDS-PAGE and immunoblotting (B), as described in Fig. 1. In (C), cells were permeabilised and exposed for 15 minutes to incremental [Ca2+] before the addition of 10 µg/ml proteinase K. After a further 30 minutes, 2 mM PMSF was added, membranes were prepared and analysed by western blotting, as above.

View larger version (42K): [in a new window]   Fig. 3. The Ca2+ dependencies of exoytosis and for the acquisition by SNAREs of trypsin resistance are unaffected following SNAP-25 truncation by BoNT/A. Chromaffin cells, in the absence () or presence ([UNK]) of 66 nM BoNT/A (see Materials and Methods), were permeabilised and exposed for 15 minutes to a range of free [Ca2+] (MgATP was not included), then catecholamine release was assayed as described in Fig. 1; the results are plotted in (A). Cells were incubated without (B) or with (C) trypsin (100 µg/ml final concentration) for a further 30 minutes, and 2 mM PMSF added before a membrane fraction was prepared. The latter samples were boiled for 10 minutes, subjected to SDS-PAGE and analysed by western blotting under identical conditions. The amount of SNAREs remaining in the trypsin-treated cells was quantified (see Materials and Methods) using photographic exposures optimised to show the Ca2+ dependence of trypsin resistance (i.e. using photographs in which the signals from BoNT/A-treated cells had been developed for longer than shown in C). The signal intensities were quantified as in Fig. 1 for control (open symbols) and BoNT/A-poisoned cells (closed symbols) and plotted for synaptotagmin I (D), syntaxin (E), SNAP-25 (F) and synaptobrevin (G). Plotted data are representative of results obtained on at least three separate occasions.

View larger version (65K): [in a new window]   Fig. 4. The majority of the trypsin-resistant SNAREs are not in SDS-resistant complexes. Chromaffin cells were permeabilised and exposed for 15 minutes to various [Ca2+], then maintained for a further 30 minutes in the absence (A) or presence (B) of 100 µg/ml trypsin, before being harvested in the presence of 2 mM PMSF and their membranes isolated. The samples were solubilised in SDS-PAGE sample buffer and split into two; one of each was boiled for 2 minutes. Boiled and non-boiled samples were subjected to SDS-PAGE and western blotting.

View larger version (45K): [in a new window]   Fig. 5. Sbr is not required for Ca2+ to trigger changes in synaptotagmin I, SNAP-25 and syntaxin. (A) Membranes, prepared from intact control and BoNT/B-treated chromaffin cells, were subjected to SDS-PAGE followed by western blotting. (B-F) Toxin-free () and BoNT/B-treated ([UNK]) chromaffin cells were permeabilised by exposure to digitonin in KGEP, with the inclusion of incremental free [Ca2+]. Following a 15 minute interval, aliquots were removed and assayed (±s.d.; n=4) for catecholamine (B); then 100 µg/ml trypsin was added and 30 minutes later 2 mM PMSF was included and a membrane fraction prepared. The samples were boiled for 2 minutes prior to SDS-PAGE and western blotting (C); the amounts of synaptotagmin (D), syntaxin (E) and SNAP-25 (F) present in each were quantified and normalised as a percentage of the largest value in each [Ca2+] series. Plotted data are representative of results obtained on at least three separate occasions.

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