Work

Protocol for single-molecule analysis of synaptic protein complex-mediated vesicle recruitment

Year: 2025

Type: article

Abstract: Single-molecule pull-down (SIM-Pull) combined with total internal reflection fluorescence (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 (SVs). We describe steps for visualizing and quantifying SV recruitment mediated by STX1A-SNARE and RIM1-Rab3a interactions. This technique allows the examination of subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104249

Authors: Akshay Kapadia, Anne-Sophie Hafner

Institutions: Radboud University Nijmegen, Donders Institute for Brain, Cognition and Behaviour

Language: English

Topic: Cellular transport and secretion

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funder: HORIZON EUROPE Framework Programme