Imaging the assembly and disassembly kinetics of cis-SNARE complexes on native plasma membranes.
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 (alpha-SNAP) concentrations.
Bar-On
Dana
D
Department of Neurobiochemistry, Tel Aviv University, Tel Aviv 69978, Israel.
Winter
Ulrike
U
Nachliel
Esther
E
Gutman
Menachem
M
Fasshauer
Dirk
D
Lang
Thorsten
T
Ashery
Uri
U
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000737:chemistry; D016716:PC12 Cells; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry*; D013997:Time Factors
D050600:SNARE Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat