Cortical F-actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells

It has been proposed recently that the F-actin cytoskeleton organizes the relative disposition of the SNARE proteins and calcium channels that form part of the secretory machinery in chromaffin cells, a neurosecretory model. To test this idea, we used confocal microscopy do determine if DsRed-SNAP-25 microdomains, which define the final sites of exocytosis along with syntaxin-1, preferentially remain in contact with F-actin cortical structures labelled by lifeact-EGFP. A quantitative analysis showed that in cells over-expressing these constructs there is a preferential colocalization, rather than a random distribution of SNAP-25 patches. To analyze the possible interactions between these proteins, we designed FRET experiments and tested whether treatment with agents that affect F-actin mobility would modify SNAP-25 movement. The significant FRET efficiencies detected suggest that direct molecular interactions occur, whereas dynamic experiments using TIRFM revealed that attenuation of cortical F-actin movement clearly diminishes the mobility of SNAP-25 clusters. Taken together, these data can be explained by a model that associates components of the secretory machinery to the F-actin cortex through flexible links.