Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'- dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.     

The cytoplast: A unit structure in chromatophores

We followed the translocation of identifiable pigment granules in living erythrophores through normal aggregation and dispersion and observed that they always return in dispersion to the same location relative to the whole pigment complex. This is interpreted to mean that each granule occupies a fixed position within a unit structure, the cytoplast. This position is retained even though the cytoplast undergoes dramatic reversals in form from ellipsoid to spheroid and back again with each aggregation and dispersion. The major structural components of the cytoplast, besides pigment granules, are microtubules and microtrabeculae. The latter constitute an irregular lattice that is confluent with microtubules and contains the pigment granules. In aggregation, the microtrabeculae shorten and seemingly contribute to the contraction of the entire cytoplast plus pigment. In dispersion, the microtrabeculae elongate in an apparent restructuring of the ellipsoidal cytoplast. The microtubules, however, persist in the cell cortex and appear to give radial direction to the pigment motion.     

THE ROLE OF MICROTUBULES IN THE MOVEMENT OF PIGMENT GRANULES IN TELEOST MELANOPHORES

When microtubules in teleost melanophores are disrupted with antimitotic agents, colchicine, high hydrostatic pressure, low temperature, and vinblastine, the alignment and movement of the pigment granules in these cells disappear; during recovery, the return of alignment and movement corresponds in both time and space with the repolymerization of microtubules. Furthermore, analysis of nearest neighbor distances in untreated melanophores reveals that pigment granules are closely associated with microtubules. Other structures such as microfilaments, the endoplasmic reticulum, and the cytoplasmic matrix do not appear to be involved. Thus we conclude that microtubules determine the alignment and are essential for the selective movements of the pigment granules in these cells. Investigations of the mechanism of movement show that microtubules are required for both centrifugal and centripetal migrations and that they do not change in number or location during redistribution of pigment. Our results further indicate that microtubules in melanophores behave as semistable organelles as determined by investigation with colchicine and hydrostatic pressure. These observations and others rule out a push-pull mechanism based on the polymerization and depolymerization of microtubules or one which distinguishes two operationally different sets of microtubules. We propose instead that particles move by sliding along a fixed array of microtubules.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). I. Energy requirements

Erythrophores isolated from the scales of the marine teleost, Holocentrus ascensionis (Osbeck), are capable of rapidly aggregating or dispersing numerous red pigment granules within their cytoplasm by translocating them along radial paths delineated by bundles of radially oriented microtubules. Pigment translocation is accompanied by transformations in the morphology of the cytoplasmic matrix, or microtrabecular lattice (MTL), in which the pigment granules are suspended. It appears that the MTL as a whole contracts toward the cell center during aggregation, carrying the pigment granules inward along with it, and is restructured during dispersion, using the radial microtubules as guides. We examined the energy requirements of pigment migration and the accompanying MTL transformations. Cellular ATP was depleted using the specific metabolic inhibitors 2,4 dinitrophenol, NaCN and oligomycin. All three of these drugs, which inhibit oxidative phosphorylation by different mechanisms, prevent both pigment dispersion and MTL transformation to dispersed morphology, while aggregation is unaffected. Inhibitor-treated cells recover normal pigment movements and MTL morphology when inhibitor is washed out of the cells with fresh medium. Potential energy apparently is stored in the MTL by some ATP-dependent process during dispersion and is converted to kinetic energy during aggregation. The results of this study strengthen the hypothesis that the MTL, working in concert with the radial microtubules, is the vehicle for pigment translocation in the erythrophore system.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

Cytoskeleton of retinular cells from the stomatopod,Gonodactylus oerstedii: possible roles in pigment granule migration

Retinular cells of the compound eyes of stomatopods (mantis shrimps) contain screening pigment granules that migrate radially in response to light. To clarify the role of the cytoskeleton in these movements, we have performed light microscopy and ultrastructural analyses of cytoskeletal organelles in retinular cells. Rhodamine phalloidin staining indicates that filamentous actin is a component of microvillar rhabdomeres and zonula adherens between retinular cells. Ultrastructural studies reveal three populations of microtubules in retinular cells that differ in their orientations and labilities to fixation. Two of these populations are oriented longitudinally in cells: the soma microtubules, found primarily in a column in the cell soma, and the more labile palisade microtubules, which extend alongside the palisade vacuole near the rhabdomere. The third, most labile microtubule population, and filaments 9–30 nm in diameter, are oriented radially in retinular cells, some within cytoplasmic bridges that span the palisade. The radial microtubules and filaments are appropriately oriented for participating in pigment granule migration. Determination of microtubule polarities in retinular cells by decoration with endogenous tubulin indicates that palisade and soma microtubules contain subpopulations having opposite polarity orientations, as has been observed in neuronal dendrites. In contrast, neighboring pigment cells contain microtubules uniformly oriented with minus ends towards the nucleus, as has been observed in most cell types studied.     

Effects of nocodazole,a new synthetic microtubule inhibitor,on movement and spreading of mouse peritoneal macrophages

Colchicine treatment enahnces movement and suppresses spreading of mouse peritoneal macrophages. The effects of colchicine could result either from disruption of cytoplasmic microtubules or from other actions of colchicine on mammalian cell processes. Nocodazole is a new synthetic microtubule inhibitor that is structurally dissimilar to colchicine and is therefore unlikely to share with colchicine any common action besides of inhibition of microtubule assembly. Nocodazole was shown here to have activities similar to colchicine on macrophage migration and spreading. This supports the idea of a direct relationship between disruption of cytoplasmic microtubules and macrophage migration and spreading.     

Photomechanical migrations of pigment granules along the retinula cells of the crayfish

The light-dependent migrations of proximal pigment granules along the photoreceptors of the crayfish compound-eye were studied in isolated retinas and eyestalks. The extent and kinetics of movement in each direction were found quantitatively equivalent to those observed in the organ in situ. These and other features make these cells to appear as intrinsically independent pigmentary effectors, directly responsive to light. During dark adaptation (DA) the pigment migrates away from the cell nucleus and accumulates along the axon in two distinct steps. Each step constitutes half of the total distance of about 180 microns and proceeds at 0.30 micron/sec. Only prolonged metabolic impairment inhibited the first phase, while the second was blocked by hypoxia, cyanide, colchicine, and D2O. The maintenance of a full DA position was also shown to be highly dependent upon metabolism. Light incidence on DA eyes is followed by an apparently monophasic expansion of the pigment from the axon towards the perikaryl region at 0.38 micron/sec. This movement was not affected by any of the foregoing agents and seems to be a passive relaxation process. Cytochalasin B had no effect on either motion. The migration in either direction has an exponential time course and is temperature dependent. Electron microscopy revealed two separate patterns of cytoplasmic organization corresponding to the cell areas where the two phases of DA occur. In the region close to the nucleus the pigment appears irregularly scattered, whereas in the axon the granules are situated arond a thick longitudinal bundle of microtubules. These results suggest the existence of two different mechanisms of pigment granule translocation operating in two separate regions of the retinula cell.     

Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.     

The ultrastructure of contractile tubules in the heliozoon Actinophrys sol and their possible involvement in rapid axopodial contraction

We employed an improved fixation procedure for electron microscopy using ruthenium red, and found a bundle of contractile tubules inside the axopodia of the heliozoon Actinophrys sol. Upon food uptake, the tubules shorten and transform into a mass of small granules when rapid axopodial contraction occurs, suggesting that these structures are involved in the process of axopodial contraction. The relationship between transformation of the contractile tubules and accompanying disassembly of the axonemal microtubules was studied by examining the ultrastructure of the contractile tubules after disassembly of the microtubules was artificially induced by cold or colchicine treatment. Granulation of the contractile tubules was induced by cold but not by colchicine treatment. During recovery from cold treatment, granular forms of the contractile tubules became re-elongated and their initial tubular appearance was restored. These results suggest that the contractile tubules in heliozoon axopodia play a role in repetitive cytoplasmic contraction.     

Remnant motility of macrophages treated with cytochalasin B in the presence of colchicine

We have previously observed that mouse peritoneal macrophages cultured for 48 h and treated with colchicine to depolymerize cytoplasmic microtubules become ameboid and cease to migrate by gliding on the substratum. We have now found that when such cells were further exposed to both colchicine and cytochalasin B, the induced ameboid movements were reversibly inhibited. Cells treated concomitantly with both drugs did not become motionless, but exhibited a remnant motility that took the form of zeiosis (blebbing). The zeiotic blebs contained ribosomes and fibrous material, but lacked organized microfilament arrays and rarely included other cytoplasmic organelles. Zeiosis appears to be a form of surface movement independent both of cytoplasmic microtubules and of the cytochalasin-sensitive contractile system. These observations imply an additional mechanism that can reversibly alter the form of the cell.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

Coelomic Cells in the Connective Tissue Spaces of the Clitellar Epithelium of Lumbricus friendi Cognetti, 1904 (Oligochaeta): an Ultrastructural Study

The ultrastructure of the connective tissue spaces in the clitellar epithelium has been studied in the earthworm Lumbricus friendi. Four morphological types of coelomic cells are described: amoebocytes, mucocyte-like cells, pigment cells and crystal-containing cells. The amoebocytes are characterized by the presence of spherical to oval electron-dense granules, phagocytic vacuoles and numerous microtubules located in the Golgi areas. The mucocyte-like cells show the features of the mucocytes reported in enchytraeid worms (globular inclusions with filamentous and homogeneous, moderately electron-dense material, as well as a filopodous process). The pigment cells contain typical spindle-shaped osmiophilic granules, microtubules (not reported before) and glycogen particles. The crystal-containing cells show inclusions which are polygonal in section with a striated substructure (periodicity of about 4.5 nm). Apart from the mucocyte-like cells, the coelomocytes showed cytoplasmic processes attached to the basement membrane of the spaces. The possible functions of these cells are discussed and a common peritoneal origin is postulated on the base of their morphological and cytological features.     

Microtubules and the calcium-dependent regulation of rat granulosa cell steroidogenesis

The possible relationship between calcium and microtubules in the regulation of granulosa cell steroidogenesis was assessed by using agents known to alter microtubule-tubulin equilibrium together with the ionophore A23187, an antibiotic that facilitates the movement of calcium across plasma membranes. Using immunofluorescence and morphometric analysis, we determined alterations in microtubule organization and overall cell shape, respectively, in response to ionophore-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-progesterone) during 24 h of culture. In addition, the influences of colchicine and nocodazole, two agents known to induce microtubule depolymerization, and of taxol, an agent that stabilizes tubulin polymers, on calcium-dependent regulation of granulosa cell progestin production in vitro were examined. Cells cultured as controls were flattened, highly irregular in outline, and associated with a complexly organized, well-spread cytoplasmic network of microtubules. In contrast, those maintained in the presence of increasing concentrations of ionophore were progressively more circular and smooth in outline, occupied less area on the growth surface, and contained cytoplasmic arrays of microtubules considerably less extensive than those of the controls and occupying areas defined by the more regular cellular perimeters. While progestin production in the absence or presence of a submaximally stimulatory concentration of A23187 was increased by both colchicine and nocodazole, the microtubule-depolymerizing agents had little to no effect on the production of the steroids by granulosa cells maximally stimulated by the ionophore. However, both basal and ionophore-induced progestin production were unaltered by taxol except at a concentration of 10 microM in the presence of 0.25 micrograms/ml A23187.(ABSTRACT TRUNCATED AT 250 WORDS)     

Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells

The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep- etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low- Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule- microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.     

Ultrastructure of microtubules in dermal melanophores and spinal nerve of the Antarctic teleost Pagothenia borchgrevinki

Summary The antarctic teleost, Pagothenia borchgrevinki inhabits the Antarctic Ocean where the water temperature remains around -1.9° C throughout the year. Dermal melanophores of this fish respond within minutes to epinephrine and theophylline with melanosome aggregation and dispersion, respectively. Numerous cytoplasmic microtubules are present in these cells despite the low environmental temperature. In longitudinal profiles, many microtubules are twisted, beaded and sometimes even branched. In cross sections, C-, U-, S-, 6- and other irregularly shaped tubules are observed. Nocodazole partially disrupts microtubules and inhibits epinephrine-induced pigment aggregation. Pigment movements are also prevented by erythro-9-[3-(2-hydroxynonyl)] adenine. Although the participation of these incomplete microtubules in cell motility remains uncertain, the results indicate that this fish has a cold-resistant microtubule system on which melanosome movements depend. Unlike those in melanophores, microtubules in the axons of spinal nerves are of uniform thickness and often contain an electron-dense core in the center.     

Pigment movements in fish melanophores: Morphological and physiological studies

Summary The ultrastructure of the melanophores of Pterophyllum scalare was studied with respect to changes in cell shape during melanosome migration and the number and distribution of microtubules within the cell extensions. Cells were fixed with pigment fully aggregated or fully dispersed. All measurements were carried out on cross sections of cell processes, i.e. sections cut perpendicular to the long axis of the cell extensions. Cross sections of processes of melanophores with dispersed pigment are more or less ovoid in shape, and microtubules are arranged predominantly just below the cell membrane. These microtubules exhibit a relatively constant centre-to-centre spacing of about 55–65 nm. Processes of melanophores with aggregated pigment seem to be collapsed; their volume is substantially decreased but their circumference equals that of dispersed melanophores. The number of microtubules is reduced, and their regular arrangement is lost. The differences in microtubule number associated with the aggregated or dispersed state occur irrespective of the nature of the agent inducing dispersion or aggregation. In addition, apparent insertion of microtubules into the plasma membrane of the cell processes and associations of microtubules with cytoplasmic densities in the cell centre are described.The results indicate a rapid disassembly and assembly of microtubules associated with pigment movements. The possible role of microtubule associations with cell membrane and densities as sites of microtubule polymerization is briefly discussed.This work was supported by a grant from the Deutsche Forschungsgemeinschaft.     

Avian erythrocyte development: microtubules and the formation of the disk shape

The development of avian erythrocytes involves a spheroid to discoid transformation in shape. The disk shape of the young erythroid cells is dependent on the presence of microtubules in a marginal bundle in the early stages of postmitotic maturation. Disassembly of microtubules with colchicine, vincristine, sulfate or cold temperature produces the spheroidal shape. Erythrocytes which have acquired the flattened ellipsoidal shape do not alter their shape with disassembly of the microtubules. The number of microtubules decreases as cell maturation occurs. The correlation coefficient for the number of microtubular profiles in one end of erythrocytes and the concentration of ribosomes (cell age) is 0.88. Microtubules of immature erythrocytes disappear more rapidly at 0°C than do microtubules of mature cells.It is concluded that microtubules play little or no role in the maintenance of mature red cell shape; however, they play an important role in the development of the flat discoid shape of avian erythrocytes during maturation.