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 adh ered to glass. The layer contained abundant filamentous actin (F-actin) loc ally 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 detectabl y, while two-thirds moved randomly; the average diffusion coefficient was 2 3 x 10(-4) mu m(2)/s. A small minority (<3%) moved rapidly and in a directe d fashion over distances more than a micron. Staining of F-actin suggests t hat such movement occurred along actin bundles. The seemingly random moveme nt of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intr acellular Mg2+ and replacing ATP with AMP-PNP. Mobility was blocked also wh en F-actin was stabilized with phalloidin, and was diminished when the acti n cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limi ted by the actin cortex. Opposing actions of the actin cortex on mobility m ay explain why its degradation has variable effects on secretion.