INTRODUCTION Intracellular transport occurs in all eukaryotic cells. Force pro- duction for transport may be provided by motor proteins moving along microtubules or actin filament tracks, or by assembly and disassembly of cytoskeletal components (see Wadsworth, 1993; Cramer et al., 1994, for reviews). Intracel- lular transport is often regulated in time and space by messenger molecules that coordinate motile events with physiological demands. The regulatory mechanisms for intra- cellular transport are only beginning to be understood. Our laboratory has been using pigment granule migration in the teleost retinal pigment epithelium (RPE) as a model with which to study the regulation and mechanisms of force pro- duction for intracellular particle transport (Burnside et al., 1982; Bruenner and Burnside, 1986; Garcia and Burnside, 1994; King-Smith et al., 1995). In the RPE of teleosts and amphibians, melanin pigment granules migrate into and out of the RPE cells’ long apical projections in response to changes in light condition (see Burnside and Nagle, 1983, for review). Pigment granules disperse into apical projections in the light and aggregate to the base of the RPE cells in the dark. Since the apical projections interdigitate with rod photoreceptor outer segments, pigment dispersion reduces the extent of rod pho- topigment bleach in bright light (Back et al., 1965). Although we do not yet understand the mechanism by which light regulates pigment granule position in RPE cells, several observations suggest that some extracellular signal from the retina is required, and that cAMP plays an important role in the intracellular signaling pathway. In the absence of retina, light has no effect on RPE pigment position in isolated RPE sheets or dissociated RPE cells (Bruenner and Burnside, 1986). However, treatments that elevate cAMP induce RPE pigment granule aggregation both in vivo and in isolated RPE preparations (Burnside et al., 1982; Burnside and Basinger, 1983; Bruenner and Burnside, 1986). Intracellular cAMP may be elevated either by stimulating endogenous adenylate cyclase with forskolin or by applying exogenous nonderiva- tized cAMP (Garcia and Burnside, 1994); both trigger pigment granule aggregation in isolated teleost RPE sheets and cells (Bruenner and Burnside, 1986; Garcia and Burnside, 1994). 33 Journal of Cell Science 109, 33-43 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 JCS7002 In the eyes of teleosts and amphibians, melanin pigment granules of the retinal pigment epithelium (RPE) migrate in response to changes in light conditions. In the light, pigment granules disperse into the cells’ long apical pro- jections, thereby shielding the rod photoreceptor outer segments and reducing their extent of bleach. In darkness, pigment granules aggregate towards the base of the RPE cells. In vitro, RPE pigment granule aggregation can be induced by application of nonderivatized cAMP, and pigment granule dispersion can be induced by cAMP washout. In previous studies based on RPE-retina co- cultures, extracellular calcium was found to influence pigment granule migration. To examine the role of calcium in regulation of RPE pigment granule migration in the absence of retinal influences, we have used isolated RPE sheets and dissociated, cultured RPE cells. Under these conditions depletion of extracellular or intracellular calcium ([Ca 2+ ] o , [Ca 2+ ] i ) had no effect on RPE pigment granule aggregation or dispersion. Using the intracellular calcium dye fura-2 and a new dye, fura-pe3, to monitor calcium dynamics in isolated RPE cells, we found that [Ca 2+ ] i did not change from basal levels when pigment granule aggregation was triggered by cAMP, or dispersion was triggered by cAMP washout. Also, no change in [Ca 2+ ] i was detected when dispersion was triggered by cAMP washout in the presence of 10 μM dopamine, a treatment previously shown to enhance dispersion. In addition, elevation of [Ca 2+ ] i by addition of ionomycin neither triggered pigment movements, nor interfered with pigment granule motility elicited by cAMP addition or washout. Since other studies have indicated that actin plays a role in both pigment granule dispersion and aggregation in RPE, our findings suggest that RPE pigment granule migration depends on an actin-based motility system that is not directly regulated by calcium. Key words: RPE, Pigment granule migration, Calcium, Fura-pe3 SUMMARY Calcium-independent regulation of pigment granule aggregation and dispersion in teleost retinal pigment epithelial cells Christina King-Smith, Paul Chen, Dana Garcia*, Homero Rey and Beth Burnside † Department of Molecular and Cell Biology, 335 Life Sciences Addition, University of California, Berkeley, CA 94720, USA *Present address: Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666-4616, USA † Author for correspondence