Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.     

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.     

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.     

Does the Introduction of a New Player,the Endoplasmic Reticulum,Create More or Less Confusion in Understanding the Mechanism(s) of Pigmentary Organelle Translocations?

In 1925, Wilson listed, in his classic third edition of Cell in Development and Heredity, four theories for the morphological and physiological characteristics of cytoplasm; each theory provided some sort of explanation as to the mechanism(s) of organelle translocations. During the past twenty years, cell biologists have focused their attentions on the cell's cytoskeleton, microtrabecular lattice, and associated mechanochemical motors which drive organelles along cytoskeletal tracks. A number of cell types have been used to study organelle translocations, but chromatophores, pigment cells, from cold-blooded vertebrates have been one of the more popular models. This article reviews some of the research findings during the past twenty years, particularly those involving cytoplasmic elements: i.e, microfilaments, intermediate filaments, microtubules, and mechanochemical motors. In addition, it contrasts the proposed involvement of these elements in organelle translocations with the endoplasmic reticulum, a tubulovesicular organelle, which we recently demonstrated is responsible, through its elongation or retraction, for the translocations of carotenoid droplets in goldfish xanthophores and swordtail fish erythrophores. Here, the carotenoid droplets are not free in the cytoplasm and do not translocate via cytoskeletal tracks, but instead are attached to or are a part of the endoplasmic reticulum. On the other hand, carotenoid droplets of squirrel fish erythrophores are free in the cytoplasm and appear to translocate via microtubules. Finally, the rates of pigmentary organelle translocations are reviewed in light of the participation of the cytoskeletal elements with the endoplasmic reticulum.     

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.     

Ultrastructural Immunogold Localization of Some Organelle-Transport Relevant Proteins in Wholemounted Permeabilized Nonextracted Goldfish Xanthophores

By whole-cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against β-tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with antiactin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti-p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti-PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti-MAP 1C (dynein) immunolabeling was generally seen on club-shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti-MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.     

Does the introduction of a new player, the endoplasmic reticulum, create more or less confusion in understanding the mechanism(s) of pigmentary organelle translocations?

In 1925, Wilson listed, in his classic third edition of Cell in Development and Heredity, four theories for the morphological and physiological characteristics of cytoplasm; each theory provided some sort of explanation as to the mechanism(s) of organelle translocations. During the past twenty years, cell biologists have focused their attentions on the cell's cytoskeleton, microtrabecular lattice, and associated mechanochemical motors which drive organelles along cytoskeletal tracks. A number of cell types have been used to study organelle translocations, but chromatophores, pigment cells, from cold-blooded vertebrates have been one of the more popular models. This article reviews some of the research findings during the past twenty years, particularly those involving cytoplasmic elements: i.e, microfilaments, intermediate filaments, microtubules, and mechanochemical motors. In addition, it contrasts the proposed involvement of these elements in organelle translocations with the endoplasmic reticulum, a tubulovesicular organelle, which we recently demonstrated is responsible, through its elongation or retraction, for the translocations of carotenoid droplets in goldfish xanthophores and swordtail fish erythrophores. Here, the carotenoid droplets are not free in the cytoplasm and do not translocate via cytoskeletal tracks, but instead are attached to or are a part of the endoplasmic reticulum. On the other hand, carotenoid droplets of squirrel fish erythrophores are free in the cytoplasm and appear to translocate via microtubules. Finally, the rates of pigmentary organelle translocations are reviewed in light of the participation of the cytoskeletal elements with the endoplasmic reticulum.     

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.     

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.     

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.     

Sexual dimorphism of motorneurons: Timbal muscle innervation in male periodical cicadas and homologous structures in females

Summary Treatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled, Later, the microfilament bundles elongate (grow), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.     

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.     

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.     

Protonic and cationic changes during cyclical cation transport driven by electron transfer

A method is described for the subcellular fractionation of goldfish xanthophores. The procedure produces relatively pure fractions of caroteniod droplets, pterinosomes, cytosol and what appears to be plasma membrane. The presence of a distinct pattern of proteins is shown to be associated with the carotenoid droplets. Treatment of the xanthophores with ACTH affects the buoyant density of some carotenoid droplets and stimulates the phosphorylation of a polypeptide associated with the carotenoid droplets.     

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.     

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.     

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC- 12 cells

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.     

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.     

In vitro reconstitution of fish melanophore pigment aggregation

Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.     

Hormone-induced dispersion or aggregation of carotenoid-containing smooth endoplasmic reticulum in cultured xanthophores from the goldfish, Carrassius auratus L.

A novel organelle movement is described in goldfish xanthophores. Smooth endoplasmic reticulum, containing carotenoids pigments, undergo MSH or c-AMP-induced dispersion. This dispersion is independent of RNA and protein synthesis, not modulated by c-GMP and inhibited by cytochalasin B but only at the relatively high concentration of 10 mug/ml. Epinephrine induces aggregation, a process that is inhibited by colchicine. These results suggest, but do not prove, the involvement of microfilaments in dispersion and microtubules in the aggregation of carotenoid-containing endoplasmic reticulum.