Protocol for studying membrane protein dynamics and associated synaptic vesicle recruitment on native membrane sheets

Plasma membrane sheets generated by controlled mechanical disruption provide direct access to the cytosolic face of the plasma membrane while preserving the native organization of membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived plasma membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We further describe their application for live and fixed imaging of membrane protein localization, organization, conformational dynamics, and protein-protein interactions, as well as quantitative membrane-associated synaptic vesicle recruitment assays. This versatile platform preserves the native membrane environment while enabling direct visualization and quantitative analysis of membrane-associated processes at high spatial resolution. The protocol can be readily adapted to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across a wide range of cellular systems. HighlightsO_LIPreparation of plasma membrane sheets exposing the cytosolic membrane surface C_LIO_LIOptimization and validation of membrane sheets from cultured cells, neurons, and isolated synaptosomes C_LIO_LIAdaptable platform for studying membrane protein interactions in native membranes C_LIO_LIQuantitative assay for membrane-associated synaptic vesicle recruitment C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/736009v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@e725a0org.highwire.dtl.DTLVardef@1576963org.highwire.dtl.DTLVardef@1c7ea73org.highwire.dtl.DTLVardef@1f13e94_HPS_FORMAT_FIGEXP M_FIG C_FIG