Migration Of Pigment Granules Research Articles

Biological Sunglasses in a Deep-Sea Squid: Pigment Migration in the Retina of Gonatus onyx.

The outward migration of ommin pigment granules from the bases to the tips of the photoreceptors in response to light has been reported in the retina of several (mostly coastal) squid species. Following exposure to light and then dark conditions, we collected and processed retinal tissue from juvenile specimens of a deep-sea oegopsid squid, Gonatus onyx. We aimed to determine whether the ommin pigment returns to baseline, and to investigate the presence of glutamate neurotransmitter signaling under both dark and light conditions. We confirmed the presence of ommin granules but observed variability in the return of pigment to the basal layer in dark conditions, as well as changes in glutamate distribution. These findings provide support for the migration of retinal ommin pigment granules as a mechanism for regulating incoming light.

Послеоперационная воспалительная реакция при выполнении эндоскопической лазерной циклодеструкции в различных режимах (экспериментальное исследование)

The study and objectification of the degree of intensity of the postoperative inflammatory response, in our opinion, is of great importance after endoscopic laser cyclodestruction (ELCD), which is the most often performed in combination with cataract surgery, when it is usually about eyes with prospects for high visual functions. Features of morphological changes and to conduct an objective analysis of the severity of the postoperative inflammatory response after ELCD, accompanied by the POP-effect and without it, was the goal of the study. The experiment carried out on rabbits in two modes (on different eyes of each animal). In the first variant, surgical intervention performed to visual response obtained in the form of coagulation of processes with a power in the diapason from 0.3 to 0.5 W. In the second variant, until the visual realization of the biological vaporization effect in the diapason from 0.9 to 1.2 W. OCT of the anterior segment with prevailing coverage of the anterior chamber in 1, 7 and 14 days. The resulting frames standardized in size. They analyzed in the programm ImageJ. Series of semithin sections were made on an Ultracut E ultramicrotome (Reicher, Austria); photos were taken on an Axio A1 light microscope (Zaiss, Germany) with an AxioCam MRc5 camera (Zaiss, Germany). During the comparison of the groups, no statistically significant differences were found between them on the corresponding days of observation (p = 0.24). Light microscopy shows extensive damage to the epithelium of the processes with the migration of pigment granules outside the tissue, however the structure of the stroma remains ordered with the preservation of vascularization. Keywords: endoscopic laser cyclodestruction, postoperative inflammation, glaucoma

Особенности морфологических изменений и послеоперационного воспалительного ответа при выполнении эндоскопической лазерной циклодеструкции в различных режимах (экспериментальное исследование)

The study and objectification of the degree of intensity of the postoperative inflammatory response, in our opinion, is of great importance after endoscopic laser cyclodestruction (ELCD), which is the most often performed in combination with cataract surgery, when it is usually about eyes with prospects for high visual functions. Features of morphological changes and to conduct an objective analysis of the severity of the postoperative inflammatory response after ELCD, accompanied by the POP-effect and without it, was the goal of the study. The experiment carried out on rabbits in two modes (on different eyes of each animal). In the first variant, surgical intervention performed to visual response obtained in the form of coagulation of processes with a power in the diapason from 0.3 to 0.5 W. In the second variant, until the visual realization of the biological vaporization effect in the diapason from 0.9 to 1.2 W. OCT of the anterior segment with prevailing coverage of the anterior chamber in 1, 7 and 14 days. The resulting frames standardized in size. They analyzed in the programm ImageJ. Series of semithin sections were made on an Ultracut E ultramicrotome (Reicher, Austria); photos were taken on an Axio A1 light microscope (Zaiss, Germany) with an AxioCam MRc5 camera (Zaiss, Germany). During the comparison of the groups, no statistically significant differences were found between them on the corresponding days of observation (p = 0.24). Light microscopy shows extensive damage to the epithelium of the processes with the migration of pigment granules outside the tissue, however the structure of the stroma remains ordered with the preservation of vascularization. Keywords: endoscopic laser cyclodestruction, postoperative inflammation, glaucoma

The role of ABCC4 in pigment granule migration in RPE of mice

ABCC4 is an ATP‐binding cassette protein that exports many substrates, including cyclic adenosine monophosphate (cAMP). In mammals, cyclic AMP accumulates in retinal photoreceptors in dark conditions. We conjecture that cyclic AMP is exported from the photoreceptors into the sub‐retinal space, where it becomes available for uptake by the retinal pigment epithelium (RPE), activating the process of pigment granule aggregation. We hypothesize that ABCC4 in photoreceptors mediates cAMP‐export and therefore is essential to the process of pigment granule migration in the RPE of mice. To investigate the involvement of ABCC4 in pigment granule movement, pigment granule density was measured along the basal part of the RPE for both wildtype and Abcc4 knockout mice that were light‐ or dark‐adapted (N = 3 for each treatment). Dark‐adapted wildtype mice showed significantly higher pigment granule density compared to light‐adapted wildtype mice, and no significant difference was seen between light‐adapted and dark‐adapted Abcc4knockout mice. A polyclonal antibody to ABCC4 used on zebrafish retinal sections showed the presence of ABCC4 in the outer retina by immunohistochemistry. These results are consistent with the hypothesis that ABCC4 plays a role in the regulating pigment granule position in RPE of mice.

Protein kinase A regulation of pigment granule motility in retinal pigment epithelial cells from fish, Lepomis spp.

Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.

A Role for ABCC4 in Regulating Pigment Granule Aggregation in Mice

ABCC4 is member of the family of ATP‐binding cassette proteins that specialize in transporting a variety of molecules out of the cell. ABCC4 exports signaling molecules present in the retina, including cyclic adenosine monophosphate (cyclic AMP). In fish, cyclic AMP regulates processes in the retinal pigment epithelium (RPE), such as dark‐adaptive pigment granule aggregation. In mammals, cyclic AMP accumulates in photoreceptors in the dark. If cyclic AMP were exported from the photoreceptors into the sub‐retinal space, it could then be available for uptake by the RPE, in which it could activate processes that drive pigment granule aggregation. We hypothesize that ABCC4 in photoreceptors mediates cAMP‐export and is needed for pigment granule migration in mice. To investigate ABCC4's involvement in pigment granule aggregation in mice, pigment granule density was measured along the basal microvilli in the RPE in both wildtype (N=3) and Abcc4 knockout mice (N=3). Pigment granule densities measured in the RPE of wild type, dark‐adapted mice differed significantly from those measured in wildtype, light‐adapted mice (p‐value = 0.02). In contrast, pigment granule density was not statistically significantly different between Abcc4 knockout mice that had been dark‐adapted or light‐adapted (p‐value = 0.13). These results support the hypothesis that ABCC4 is involved in the process of pigment granule migration in mice and are consistent with a novel role for cyclic AMP as a cell‐to cell signaling molecule in the retina.Support or Funding InformationThis project was supported by Texas State University in the form of an undergraduate research fellowship to AMP and funds from the Office of the Provost awarded to DMG in recognition of her service as Presidential Fellow. DEI received salary support from HSI STEM Program (84.031c) Award #P031C160035.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ectoplasm, ghost in the R cell machine?

Drosophila photoreceptors (R cells) are an extreme instance of sensory membrane amplification via apical microvilli, a widely deployed and deeply conserved operation of polarized epithelial cells. Developmental rotation of R cell apices aligns rhabdomere microvilli across the optical axis and enables enormous membrane expansion in a new, proximal distal dimension. R cell ectoplasm, the specialized cortical cytoplasm abutting the rhabdomere is likewise enormously amplified. Ectoplasm is dominated by the actin-rich terminal web, a conserved operational domain of the ancient vesicle-transport motor, Myosin V. R cells harness Myosin V to move two distinct cargoes, the biosynthetic traffic that builds the rhabdomere during development, and the migration of pigment granules that mediates the adaptive "longitudinal pupil" in adults, using two distinct Rab proteins. Ectoplasm further shapes a distinct cortical endosome compartment, the subrhabdomeral cisterna (SRC), vital to normal cell function. Reticulon, a protein that promotes endomembrane curvature, marks the SRC. R cell visual arrestin 2 (Arr2) is predominantly cytoplasmic in dark-adapted photoreceptors but on illumination it translocates to the rhabdomere, where it quenches ongoing photosignaling by binding to activated metarhodopsin. Arr2 translocation is "powered" by diffusion; a motor is not required to move Arr2 and ectoplasm does not obstruct its rapid diffusion to the rhabdomere.

Calcium-Activated Myosin V Closes the Drosophila Pupil

Approximately 40 years ago, an elegant automatic-gain control was revealed in compound eye photoreceptors: In bright light, an assembly of small pigment granules migrates to the cytoplasmic face of the photosensitive membrane organelle, the rhabdomere, where they attenuate waveguide propagation along the rhabdomere. This migration results in a "longitudinal pupil" that reduces rhodopsin exposure by a factor of 0.8 log units. Light-induced elevation of cytosolic free Ca(2+) triggers the migration of pigment granules, and pigment granules fail to migrate in a mutant deficient in photoactivated TRP calcium channels. However, the mechanism that moves photoreceptor pigment granules remains elusive. Are the granules actively pulled toward the rhabdomere upon light, or are they instead actively pulled into the cytoplasm in the absence of light? Here we show that Ca(2+)-activated Myosin V (MyoV) pulls pigment granules to the rhabdomere. Thus, one of MyoV's several functions is also as a sensory-adaptation motor. In vitro, Ca(2+) both activates and inhibits MyoV motility; in vivo, its role is undetermined. This first demonstration of an in vivo role for Ca(2+) in MyoV activity shows that in Drosophila photoreceptors, Ca(2+) stimulates MyoV motility.

Calcium movements during pigment aggregation in freshwater shrimp chromatophores

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.

Time course and development of light adaptation processes in the outer zebrafish retina

Retinomotor movements are morphological changes in the outer retina in response to changing light conditions. They can be separated into two components: Migration of pigment granules within the microvilli of the retinal pigment epithelium (RPE) and positional changes in photoreceptor cells. These positional changes optimize exposure of the cone and rod photoreceptors to light. The aim of this study was to analyze both the time course of retinomotor movements in the adult zebrafish and the maturation of these processes in the developing fish. We show that retinomotor movements are used as a dark/light adaptation mechanism in zebrafish. In adult zebrafish, melanin granules of the RPE migrate with constant speed and reach the fully light adapted (LA) state approximately after 1 h. In contrast, about two thirds of double cone outer segment movements are finished in 5 min, and are fully completed in 10 to 20 min. During development there are three crucial stages leading to mature retinomotor movements in response to light: at 5 dpf (days post fertilization) the migration of pigment granules begins, at 20 dpf the pigment granules condense in the apical part of the RPE microvilli, and at 28 dpf, concomitant with the functional maturation of rods, the double cones contract as in adult retinas.

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

BackgroundIn fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents.ResultsThe general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion.ConclusionsOur results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Rhythmicity of the Pigments in the Compound Eye of Daphnia longispina (Cladocera)

ABSTRACT We investigated pigment granule migration in the eye of the crustacean, Daphnia longispina. Pigment granule positions were indirectly observed by monitoring the pigment granule surface area (PGSA) under constant illumination as well as under natural lighting conditions. Significant variations were observed in the PGSA during the course of the 24-h night-day cycle. These phenomena, which persisted under continuous illumination, suggest that an endogenous mechanism is involved in bringing about the observed diurnal morphological changes in the compound eye of Daphnia longispina.

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Melatonin receptor RNA expression in Xenopus retina

Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse-transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

Nitric oxide synthase and background adaptation in Xenopus laevis

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by α-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). α-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain. In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background. Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3′,5′ guanosine monophosphate (cGMP)-accumulating fibers in the PI. The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.

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.

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

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

Light-dependent pigment migration in blowfly photoreceptors studied by in vivo CLSM

The light-dependent migration of pigment granules in the soma of fly photoreceptors has been studied in vivo with a fast confocal laser scanning microscope. Images as well as photometric measurements were obtained in the reflection and fluorescence modes. Measurements at the single cell level were performed by using water immersion. The illumination of dark adapted photoreceptors causes a rapid increase in reflectance due to the migration of light scattering pigment granules toward the rhabdomeres. In the steady-state, the reflection signal strongly fluctuates, indicating that the pigment granules undergo a very rapid fluctuating movements. A major part of the reflection signal is due to light back-scattered by the pigment granules and channeled through the light guiding rhabdomeres. The optical axes of the rhabdomeres can thus be directly traced and appear to be directed toward the optical centre of the corresponding facet lens. Simultaneous with the reflection increase, the fluorescence of the photoreceptors decreases, because the pigment granules accumulating near the rhabdomeres act as a light-controlling pupil. Broad-band, white light filtered by the predominantly blue absorbing pupil causes an increased fraction of the visual pigment in the rhodopsin state.

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.