Membrane-directed molecular assembly of the neuronal SNARE complex.
Bottom Line: Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution.Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study.Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted.
Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.Show MeSH
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Mentions: To observe physiological SNARE complexes on mica for AFM studies, freshly cleaved mica disks were placed in a fluid chamber. Two hundred microlitres of imaging buffer solution containing 140 mM NaCl, 10 mM HEPES and 1 mM CaCl2, was placed at the centre of the cleaved mica disk. Prior to deposition on mica, t-SNARE reconstituted vesicles were gently layered on the mica surface, and incubated at room temperature for 60 min. prior to washing. Isolated SVs were added, incubated for 20 min., prior to three washes using the imaging buffer. During repeated imaging of the SVs attached to the t-SNARE-membrane supported by mica, some SVs are dislodged by the AFM cantilever tip, exposing the t-/v-SNARE ring complex (Fig. 5D).
Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.