Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.
In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.
Lang
T
T
Max-Planck-Institut für biophysikalische Chemie, D-37077 Göttingen, Germany.
Wacker
I
I
Wunderlich
I
I
Rohrbach
A
A
Giese
G
G
Soldati
T
T
Almers
W
W
D000199:Actins / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000502:physiology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology*; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis; D008274:Magnesium / Q000502:physiology; D009068:Movement; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C004717:diacetylmonoxime; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D003931:Diacetyl