Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.     

Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.     

Regulation of pigment organelle translocation. I. Phosphorylation of the organelle-associated protein p57

Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57.     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.     

Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish

We reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr approximately 125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.     

In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.     

Regulation of organelle transport: Lessons from color change in fish

Organelles transported along microtubules are normally moved to precise locations within cells. For example, synaptic vesiceles are transported to the neruronal synapse, the Golgi apparatus is generally found in a perinuclear location, and the membranes of the endoplasmic reticulum are actively extended to the cell periphery. The correct positioning of these organelles depends on microtubules and microtubule motors. Melanophores provide an extreme example of organized organelle transport. These cells are specialized to transport pigment granules, which are coordinately moved towards or away from the cell center, and result in the cell appearing alternately light or dark. Melanophores have proved to be an ideal system for studying the mechanisms by which the cell controls the direction of its organelle transport. Pigment granule dispersion (the movement away from the cell center) requires protein phosphorylation, while pigment aggregation (the movement towards the cell center) requires protein dephosphorylation. The target of this phosphorylation and dephosphorylation event is a protein that interacts with the microtubule motor protein, kinesin. Thus, the direction of organelle transport along microtubules may be regulated by controlling the activity of a microtubule motor.     

Ultrastructural demonstration of hormone-induced movement of carotenoid droplets and endoplasmic reticulum in xanthophores of the goldfish,Carassius auratus L.

Summary The hormone-induced pigment dispersion in primary cultures of xanthophores of goldfish (Carassius auratus L.) has been shown to involve the dispersion of not only carotenoid droplets but also of smooth endoplasmic reticulum. The dispersion of these organelles is inhibited by cytochalasin B and is accompanied by thinning of the cell body, thickening of the processes, and also overall changes in cellular morphology (process extension) under certain conditions. Electron microscopic examination of heavy meromyosin treated glycerinated xanthophores in scales revealed the presence of actin filaments in these cells.This work was supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S.     

Molecular mechanisms of pigment transport in melanophores.

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.     

Molecular Mechanisms of Pigment Transport in Melanophores

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

Protein phosphorylation and the two stages of pigment organelle dispersion in permeabilized xanthophores: organelle protein phosphorylation alone supports only the first stage

We reported previously that, in cultured goldfish xanthophores, dispersion of aggregated carotenoid droplets (CDs) requires the specific phosphorylation of the CD protein p57 by a cAMP-dependent protein kinase and the presence of cytosol. We report here that, in permeabilized cells, the addition of the catalytic subunit of cAMP-dependent protein kinase and ATP phosphorylates p57 and converts the CDs from an immobile to a mobile state (first stage of CD dispersion). However, the CDs are restricted to the vicinity of the original site of the CD aggregate and do not actually disperse (second stage of CD dispersion) unless cytosol is also added. We propose that this process may be related to aspects of secretory processes.     

Tumor necrosis factor induces hyperphosphorylation of kinesin light chain and inhibits kinesin-mediated transport of mitochondria

The molecular motor kinesin is an ATPase that mediates plus end-directed transport of organelles along microtubules. Although the biochemical properties of kinesin are extensively studied, conclusive data on regulation of kinesin-mediated transport are largely lacking. Previously, we showed that the proinflammatory cytokine tumor necrosis factor induces perinuclear clustering of mitochondria. Here, we show that tumor necrosis factor impairs kinesin motor activity and hyperphosphorylates kinesin light chain through activation of two putative kinesin light chain kinases. Inactivation of kinesin, hyperphosphorylation of kinesin light chain, and perinuclear clustering of mitochondria exhibit the same p38 mitogen-activated kinase dependence, indicating their functional relationship. These data provide evidence for direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways.     

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Unusual leucophore-like cells specifically appear in the lineage of melanophores in the periodic albino mutant of Xenopus laevis

In the periodic albino mutant (a(p)/a(p)) of Xenopus laevis, peculiar leucophore-like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild-type (+/+). These leucophore-like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of alpha-melanocyte stimulating hormone (alphaMSH), and (4) containing an abundance of bizarre-shaped, reflecting platelet-like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore-like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to alphaMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore-like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore-like cells are essentially similar to melanophores of the wild-type with respect to their localization in the skin and manner of response to alphaMSH, (3) the leucophore-like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet-like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore-like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

MLN64 is involved in actin-mediated dynamics of late endocytic organelles

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.