In Vitro Reconstitution of Chromaffin Granule–Cytoskeleton Interactions: Ionic Factors Influencing the Association of F‐Actin With Purified Chromaffin Granule Membranes

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Abstract
Chromaffin granules are the secretory vesicles directly involved in exocytosis of catecholamines, enkephalins, and other components from adrenal medullary cells. The granules occupy a large portion of the cytoplasmic volume and thus may interact extensively with cytoskeletal elements such as actin. Indeed, using both sedimentation techniques and falling ball viscometry [Fowler et al: J Cell Biol 88:388, 1981] to measure actin binding by membranes, we were able to show that chromaffin granules bind F‐actin via a protein site on the membrane, and that these interactions are reversibly inhibited by raising the free calcium ion concentration to micromolar levels ([Ca++]free for half‐maximal inhibition approximately 2.6 × 10−7M)[Fowler and Pollard: Nature 295:336, 1982]. Here, we show that F‐actin‐chromaffin granule interactions are unaffected by changes in pH between about pH 6.4 and 7.4 but are about 50% inhibited by raising the pH from 7.5 to 8.0. They are also 50% inhibited by increasing the KCl concentration to about 200 mM but are not significantly affected by increasing concentrations of K‐glutamate up to 500 mM or by varying the MgCl2 concentration between 0 and 6 mM. The interactions between chromaffin granule membranes and F‐actin are also reduced in the presence of AIP, AMP‐PNP, or free pyrophosphate; cAMP and AMP are without effect.The ability of chromaffin granule membranes to interact with F‐actin under conditions that may approximate the resting intracellular environment (neutral pH, low KCl, 1–2 mM MgCl2, 1 mM ATP, [Ca++]free < 10−7M, 30°C) suggests that these interactions may partially reconstitute naturally occurring associations between chromaffin granules and the cytoskeleton. Further, regulation of chromaffin granule membrane‐actin interactions by ionic factors (pH, calcium, chloride ions, nucleotides) that could vary intracellularly leads us to propose that associations between actin and the chromaffin granule membrane could influence the location and dislocation of these organelles in the cytoplasm.