Dynamics of immature secretory granules: Role of cytoskeletal elements during transport, cortical restriction, and F-actin-dependent tethering

Secretory granules store neuropeptides and hormones and exhibit regulated e xocytosis upon appropriate cellular stimulation. They axe generated in the trans-Golgi network as immature secretory granules, short-lived vesicular i ntermediates, which undergo a complex and poorly understood maturation proc ess. Due to their short half-life and low abundance, real-time studies of i mmature secretory granules have not been previously possible. We describe h ere a pulse/chase-like system based on the expression of a human chromogran in B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynam ics during maturation. Live cell imaging revealed that newly formed immatur e secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that th e cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move random ly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late p hase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.