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.     

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.     

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.     

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.     

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.     

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.     

On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue

On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.     

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.     

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.     

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.     

A new look at the cellular scaffold by embedment-free electron microscopy method

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.     

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.     

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.     

Adhesion of intermediate filaments and lipid droplets in adrenal cells studied by field emission scanning electron microscopy

High-resolution field emission scanning electron microscopy was used to study the organisation of intermediate filaments around lipid droplets and their binding to these droplets, in primary culture of bovine adrenal cells. Whole-mount preparations of intermediate filaments and bound lipid droplets were prepared from cells grown on Formvar-coated grids and processed by freeze-drying. Intermediate filaments were seen as an interconnected network enveloping the entire droplet. The bound filaments appear to be directly adherent to the surface of the droplet and hence take on its curved contour. The binding of the filaments to the droplets was determined by means of tilting. This study provides a new approach to investigate the cytoskeleton and its associated structures with high-resolution three-dimensional images.     

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.     

Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.     

The localization of estrogen receptors on human osteoblasts

Attempts to identify estrogen target cells in bone by immunocytochemistry using antibodies to the receptor have proved to be controversial. The aim of this study was therefore to determine whether immunogold labeling can be used as a technique for the localization of estrogen receptors (ER) on a human osteoblast-like cell line. The aim was also to determine the distribution of ER on the cell surface by using a scanning electron microscope (SEM) and intracellularly by using a transmission electron microscope (TEM). Labeling of the cytoplasmic material was seen around areas that appeared to be a disrupted plasma membrane. No nuclear or perinuclear labeling could be detected. The conclusion can be made that SEM immunogold labeling combined with TEM can be regarded as a practical technique for localizing ER on human osteoblasts. This article clearly demonstrated that osteoblast-like cells do express ER at low levels and that, although cytoplasmic immunoreactivity could be detected, no, nuclear or perinuclear labeling was found.     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金所特异标记。本文结果表明,结合于核孔复合体上     

Ultrastructural immunogold labeling of glial filaments in osmicated and unosmicated epoxy-embedded tissue

On-grid (post-embedding) immunolabeling methods with epoxy resins have been difficult to apply to thin structures such as intermediate filaments, which may remain inaccessible within the plastic. In this study, glial fibrillary acidic protein (GFAP), the major protein of astrocyte intermediate filaments, was localized with a post-embedding immunogold method, using both unosmicated and osmicated material embedded in epoxy resin. The tissue studied was from a diagnostic brain biopsy on a child with Alexander's disease. This disorder is characterized by proliferation of astrocyte intermediate filaments and formation of Rosenthal fibers. With unosmicated tissue, as in a previous study, extensive labeling of the glial filaments was achieved only when ultra-thin sections were pre-treated with dilute sodium ethoxide, an agent that dissolves plastic. Fifteen-nm gold could be used. With osmicated tissue, localization to glial filaments required pre-treatment with sodium ethoxide and with the oxidizing agent sodium metaperiodate, followed by the use of small (5 nm) colloidal gold. That 5-nm gold was required for labeling filaments in osmicated material suggested that osmication increases problems of penetrability and antigen accessibility within ultra-thin sections. The large Rosenthal fibers were labeled by 15-nm gold in both unosmicated and osmicated material. The methods employed may be useful for electron immunolocalizations to other thin structures in material embedded in epoxy resin.     

The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol—the substrate for the first enzyme, namely P450_scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca²⁺/calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca²⁺/calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cylic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca²⁺/calmodulin possibly by redistributing cellular Ca²⁺. Ca²⁺/calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomoyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca²⁺/calmodulin) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.