Syntaxin clusters and cholesterol affect the mobility of Syntaxin1a

Syntaxin1a (Syx1a) is essential for stimulated exocytosis in neuroendocrine cells. The vesicle docking process involves the formation of nanoscale Syx1a domains on the plasma membrane and the Syx1a clusters disintegrate during the fusion process. Syx1a nanodomains are static yet Syx1a molecules dynamically enter and leave the domains; the process by which these clusters maintain this balance is unclear. In this work, the dynamics of the Syx1a molecules is elucidated relative to the cluster position through a labeling strategy that allows both the bulk position of the Syx clusters to be visualized concurrent with the trajectories of single Syx1a molecules on the surface of PC12 cells. Single Syx1a molecules were tracked in time relative to cluster positions to decipher how Syx1a moves within a cluster and when clusters are not present. Syx1a is mobile on the plasma membrane, more mobile at the center of clusters, and less mobile near the edges of clusters; this depends on the presence of the N-terminal Habc domain and cholesterol, which are essential for proper exocytosis. Simulations of the dynamics observed at clusters support a model where clusters are maintained by a large cage (r = 100 nm) within which Syx1a remains highly mobile within the cluster (r = 50 nm). The depletion of cholesterol dramatically reduces the mobility of Syx1a within clusters and less so over the rest of the plasma membrane. This suggests that fluidity of Syx1a supramolecular clusters is needed for function.