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ABSTRACT: Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.
The Journal of Cell Biology 08/2012; 198(3):323-30. · 10.26 Impact Factor
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ABSTRACT: Styryl dyes are widely used to study synaptic vesicle (SV) recycling in neurons; vesicles are loaded with dye during endocytosis, and dye is subsequently released via exocytosis. During putative kiss-and-run exocytosis, efflux of dye from individual SVs has been proposed to occur via two sequential steps: dissociation from the membrane followed by permeation through a small fusion pore. To improve our understanding of the kinetics of efflux of dye from vesicles during kiss-and-run events, we examined the rates of efflux of different dyes through nanometer-scale pores formed in membranes by the toxins melittin and α-hemolysin; these pores approximate the size of fusion pores measured in neuroendocrine cells. We found that the axial diameter of each dye was a crucial determinant for permeation. Moreover, the two dyes with the largest cross-sectional areas were completely unable to pass through pores formed by a mutant α-hemolysin that has a slightly smaller pore than the wild-type toxin. The overall time constant for efflux (seconds) of each dye was orders of magnitude slower than the time constant for dissociation from membranes (milliseconds). Thus, the permeation step is rate-limiting, and this observation was further supported by atomistic molecular dynamics simulations. Together, the data reported here help provide a framework for interpreting dye destaining rates from secretory vesicles.
Biochemistry 08/2011; 50(35):7493-502. · 3.42 Impact Factor
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ABSTRACT: Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca(2+) sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca(2+) sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.
Molecular biology of the cell 05/2011; 22(13):2324-36. · 5.98 Impact Factor
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ABSTRACT: Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.
Biophysical Journal 08/2009; 97(1):101-9. · 3.65 Impact Factor
