Spatial Distribution and Dynamics of Syntaxin-1 in Live PC-12 Cells

Abstract

The soluble N-ethyl-maleimide-sensitive factor-attachment protein receptor (SNARE) proteins play an important role in vesicle fusion. The spatial distribution of these proteins in live cells is largely unknown. Clusters of syntaxin-1 (Syx-1) were observed previously by Hell, Lang and co-workers using stimulated emission depletion (STED) on PC-12 cell membrane sheets with immunostaining.1 The clusters may be docking sites for large dense-core secretory vesicles (LDCVs), which can then undergo exocytosis. In this study we transfected rat pheochromocytoma (PC-12) cells with a plasmid encoding Syx-1 tagged with the photoactivatable protein mEos2 at the N-terminus. Photoactivation and localization of single molecules was used to study the distribution of Syx-1 in live PC12 cells, while single particle tracking (SPT) was used to study the dynamics. The data indicate inhomogeneous diffusion, with some Syx-1 molecules appearing confined and others seemingly diffusing freely. A plot of is initially linear with a slope corresponding to a free diffusion constant of ∼0.15 µm2/sec. The spatial distribution is somewhat clustered, but seemingly much less so than in the earlier study of unroofed cells. LDCVs were imaged in sequence with Syx-1 using EGFP labeled tissue plasminogen activator (tPA). Preliminary data suggest that LDVCs visit the plasma membrane along specific branched paths whose locations tend to be anti-correlated with the distribution of Syx-1. 1JJ Sieber, et al. Science 317, 1072–1076 (2007).