In Situ Synthesis\nand Visualization of Membrane SNAP25\nNano-Organization

Cryo-electron tomography (cryo-ET) can provide insights\ninto the\nstructure and states of natural membrane environments to explore the\nrole of SNARE proteins at membrane fusion and understand the relationship\nbetween their subcellular localization/formation and action mechanism.\nNevertheless, the identification of individual molecules in crowded\nand low signal-to-noise ratio membrane environments remains a significant\nchallenge. In this study, cryo-ET is employed to image near-physiological\nstate 293T cell membranes, specifically utilizing in situ synthesized\ngold nanoparticles (AuNPs) bound with cysteine-rich protein tags to\nsingle-molecularly labeled synaptosomal-associated protein 25 (SNAP25)\non the membrane surface. The high-resolution images reveal that SNAP25\nis predominantly located in regions of high molecular density within\nthe cell membrane and aggregates into smaller clusters, which may\nincrease the fusion efficiency. Remarkably, a zigzag arrangement of\nSNAP25 is observed on the cell membrane. These findings provide valuable\ninsights into the functional mechanisms of SNARE proteins.