Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Abstract

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

Human Insulin Vesicle Dynamics During Pulsatile Secretion

“…Over longer time spans, especially between meals when secretion stimulation is much diminished, we expect that new (fluorescent) vesicles will repopulate the membrane-proximal pool, arriving there shortly after they bud from the Golgi body (as has been suggested for other types of endocrine cells) (50).…”

“…The rapid and directed movement of new vesicles from the Golgi body to the plasma membrane is distinct from the relatively slow and random movement of vesicles once they are near the plasma membrane (50). Of note, the average diffusion coefficient of insulin vesicles in clonal ␤-cells (36) is approximately two orders of magnitude greater than the average diffusion coefficient we report here (4.9 Ϯ 0.5 ϫ 10 Ϫ12 cm 2 /s) in primary cultures of human pancreatic ␤-cells.…”

Myrip Couples the Capture of Secretory Granules by the Actin-Rich Cell Cortex and Their Attachment to the Plasma Membrane

“…A wealth of mechanistic information has been obtained on membrane fusion, but less is known on the recruitment of SGs at the cell periphery and their attachment to the plasma membrane (PM), which is required for the formation of a pool of ready-to-fuse SGs (Verhage and Sorensen, 2008). SGs are formed at the trans-Golgi network and transported along microtubules to the cell periphery (Rudolf et al, 2001). They cannot be transferred directly from microtubules to the PM, but diffuse within the actin-rich cortex until they find an attachment site or undergo another microtubule-based run (Huet et al, 2006).…”

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

“…SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have a key role in membrane fusion (Jahn and Scheller, 2006), but little is known about upstream events (i.e., the recruitment of SGs at release sites and their docking at the plasma membrane). SGs are transported along microtubules from the trans-Golgi network to the cell periphery (Rudolf et al, 2001;Varadi et al, 2002) where they accumulate in the actin-rich cortex, a dense meshwork of filaments that restricts their mobility and is thought to hinder their access to the cell surface (Trifaro et al, 2000).…”