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 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.
Rudolf
R
R
Department of Neurobiology, Interdisciplinary Center for Neuroscience, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
Salm
T
T
Rustom
A
A
Gerdes
H H
HH
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002864:Chromogranins / Q000235:genetics / Q000378:metabolism; D045683:Furin; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D015388:Organelles / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology*; D013381:Subtilisins / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014162:Transfection
D000199:Actins; D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002864:Chromogranins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; D013381:Subtilisins; D045683:Furin; C037068:latrunculin B; D015739:Nocodazole