In Situ Synthesis and Visualization of Membrane SNAP25 Nano-Organization.

Cryo-electron tomography (cryo-ET) can provide insights into the structure and states of natural membrane environments to explore the role of SNARE proteins at membrane fusion and understand the relationship between their subcellular localization/formation and action mechanism. Nevertheless, the identification of individual molecules in crowded and low signal-to-noise ratio membrane environments remains a significant challenge. In this study, cryo-ET is employed to image near-physiological state 293T cell membranes, specifically utilizing in situ synthesized gold nanoparticles (AuNPs) bound with cysteine-rich protein tags to single-molecularly labeled synaptosomal-associated protein 25 (SNAP25) on the membrane surface. The high-resolution images reveal that SNAP25 is predominantly located in regions of high molecular density within the cell membrane and aggregates into smaller clusters, which may increase the fusion efficiency. Remarkably, a zigzag arrangement of SNAP25 is observed on the cell membrane. These findings provide valuable insights into the functional mechanisms of SNARE proteins.