Single-molecule analysis of synaptic protein complexes and vesicle recruitment
Single-molecule pull-down (SIM-Pull) combined with TIRF microscopy enables direct visualization of proteins and multi-protein complexes. Here, we present an extended SIM-Pull protocol for analyzing protein interactions at the active zone and their ability to recruit isolated synaptic vesicles (SV). SV recruitment mediated by STX1A-SNARE or RIM1-Rab3a interactions, respectively; can be directly visualized and quantified. This technique opens new avenues to examine the subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context. HighlightsO_LIExtended SIM-Pull protocol combining biochemical isolation with TIRF microscopy to study synaptic protein complexes at near-native environment C_LIO_LIEnables direct quantification of synaptic vesicle recruitment at the surface (active zone) via protein-mediated vesicle tethering C_LIO_LIAdaptable platform for probing molecular interactions of protein complexes within different neuronal, cellular or subcellular compartments C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/671146v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c42949org.highwire.dtl.DTLVardef@1727b9aorg.highwire.dtl.DTLVardef@110ec6aorg.highwire.dtl.DTLVardef@114ec7_HPS_FORMAT_FIGEXP M_FIG C_FIG Institutional permissionsAnimals were handled and maintained according to the guidelines laid down by the Animal Welfare Body (AWB) (Instantie voor Dierenwelzijn IvD) in line with the animal experimentation policy within Radboud University and RadboudUMC; under the license/protocol numbers 2021-0040-001/002 to Dr. Anne-Sophie Hafner.