title: Spatial regulation of exocytic site and vesicle mobilization by the actin cytoskeleton.
abstract: Numerous studies indicate a role for the actin cytoskeleton in secretion. Here, we have used evanescent wave and widefield fluorescence microscopy to study the involvement of the actin cytoskeleton in secretion from PC12 cells. Secretion was assayed as loss of ANF-EmGFP in widefield mode. Under control conditions, depolarization induced secretion showed two phases: an initial rapid rate of loss of vesicular cargo (tau = 1.4 s), followed by a slower, sustained drop in fluorescence (tau = 34.1 s). Pretreatment with Latrunculin A changed the kinetics to a single exponential, slightly faster than the fast component of control cells (1.2 s). Evanescent wave microscopy allowed us to examine this at the level of individual events, and revealed equivalent changes in the rates of vesicular arrival at the plasma membrane immediately following and during the sustained phase of release. Co-transfection of mCherry labeled β-actin and ANF-EmGFP demonstrated that sites of exocytosis had an inverse relationship with sites of actin enrichment. Disruption of visualized actin at the membrane resulted in the loss of specificity of exocytic site.
authors.affiliation: Department of Anesthesia, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: David A
authors.initials: DA
authors.lastname: Richards
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D047630:Depsipeptides / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D008954:Models, Biological; D016716:PC12 Cells; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D013194:Staining and Labeling; D053778:Thiazolidines / Q000494:pharmacology; D049411:Utrophin / Q000378:metabolism
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D053778:Thiazolidines; D049411:Utrophin; C057531:jasplakinolide; D049452:Green Fluorescent Proteins; D009320:Atrial Natriuretic Factor; C037067:latrunculin A