Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells.

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.

Effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility in isolated retinal pigment epithelial (RPE) cells of green sunfish, Lepomis cyanellus.

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin-disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1-1 microM), but the drug halted movement of most pigment granules and stimulated rapid bi-directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide-treated cells demonstrated slow, creeping centrifugal movements and more rapid bi-directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle-like structures, which contained F-actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide-treated cells revealed a more reticulated network of actin compared to antibody-labeled control cells. These results indicate that jasplakinolide-induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.

Bidirectional pigment granule migration in isolated retinal pigment epithelial cells requires actin but not microtubules.

In the teleost retinal pigment epithelium (RPE), melanin pigment granules disperse into long apical projections in the light and reaggregate into the cell body in the dark. To investigate the cytoskeletal mechanisms responsible for these movements, we have examined the effects of cytoskeletal inhibitors on pigment granule transport in cultured, dissociated RPE cells using time-lapse video microscopy. The kinetics of pigment granule transport during normal aggregation and dispersion are quite distinct: during aggregation, all pigment granules undergo simultaneous, nonsaltatory centripetal movement (mean velocity 3.6 microm/min); during dispersion, individual granules undergo independent, bidirectional saltatations (mean velocities 3.7 microm/min centrifugal; 1.1 microm/min centripetal). Nocodazole disruption of microtubules within the RPE apical projections had little effect on the kinetics of pigment granule movement, and essentially no effect on extent of pigment granule aggregation or dispersion, or on maintenance of the fully aggregated or fully dispersed states. In contrast, cytochalasin D (CD) treatment blocked net aggregation and dispersion of pigment granules, and compromised maintenance of the fully aggregated and dispersed states. These observations suggest that the actin cytoskeleton plays an important role in both centripetal and centrifugal transport of pigment granules in teleost RPE cells.

Calcium-independent regulation of pigment granule aggregation and dispersion in teleost retinal pigment epithelial cells.

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 projections, 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 ([Ca2+]o, [Ca2+]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 [Ca2+]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 [Ca2+]i was detected when dispersion was triggered by cAMP washout in the presence of 10 microM dopamine, a treatment previously shown to enhance dispersion. In addition, elevation of [Ca2+]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.

Pigment granule migration in crustacean photoreceptors requires calcium.

We have investigated the role of calcium in the regulation of pigment granule migration in photoreceptors of the semi-terrestrial crab, Sesarma cinereum. Isolated crab eyes (eyecup plus eyestalk) were maintained in crustacean Ringer either prepared normally or calcium-free plus 50 mM EGTA. Pigment granule movement was indirectly observed by monitoring reflectance from the eye during light stimuli using intracellular optical physiological techniques. Electroretinograms (ERGs) were also measured during light stimuli. EGTA treatment caused gradual loss of centripetal migration of pigment granules (normally leading to pupillary closure), and photoreceptors eventually became locked in the open-pupil, dark-adapted state despite repeated stimuli. In contrast, ERG responses continued throughout EGTA treatment, although the size and shape of the response was altered. Normal ERG responses and pigment granule movements returned after replacing EGTA-Ringer with normal-calcium medium. These results suggest that centripetal migration of pigment granules in crustacean photoreceptors requires calcium.

Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells.

To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.