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Imaging the assembly and disassembly kinetics of cis ‐SNARE complexes on native plasma membranes
Author(s) -
Bar-On Dana,
Winter Ulrike,
Nachliel Esther,
Gutman Menachem,
Fasshauer Dirk,
Lang Thorsten,
Ashery Uri
Publication year - 2008
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2008.08.040
Subject(s) - kinetics , biophysics , membrane , chemistry , organelle , cytoplasm , maleimide , receptor–ligand kinetics , biochemistry , microbiology and biotechnology , receptor , biology , polymer chemistry , physics , quantum mechanics
Mild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane‐biochemistry kinetics. We monitor soluble NSF‐attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two‐phase kinetics for the assembly process and dependence of the disassembly kinetics on both N ‐ethyl maleimide‐sensitive factor (NSF) and soluble NSF‐attachment protein ( α ‐SNAP) concentrations.