Alpha-actinin localization in the cleavage furrow during cytokinesis

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.     

Electron microscopic localization of cytoplasmic myosin with ferritin- labeled antibodies

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin- antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.     

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.     

Coalignment of microtubules, cytokeratin intermediate filaments, and collagen fibrils in a collagen-secreting cell system

The distribution of microtubules and intermediate filaments in the collagen-secreting scleroblasts of the goldfish scale was investigated by immunofluorescence and electron microscopy. Many of the microtubules and cytokeratin type intermediate filaments formed bundles that were aligned with the underlying, parallel collagen fibrils. The intermediate filament bundles were evenly spaced and located adjacent to the basal plasma membrane. The microtubules, on the other hand, were located further away from the membrane, although many were found very close to the intermediate filament bundles. No detectable change was observed in scleroblast microtubules when cells on scales were treated with colchicine or cooled (greater than or equal to 0 degrees C) for up to 1 h. Cells had to be cooled overnight before the microtubules were affected. The final number and length of the microtubules in the cell depended only on the final steady-state temperature and not the temperature history of the scale cell, and steady state was reached more slowly at colder temperatures. The microtubules but not the intermediate filaments rapidly (within 5 min) and reversibly depolymerized when cells were chilled to -2 approximately -4 degrees C. When chilled cells were warmed, the microtubules polymerized back, within 15 min at room temperature, to the same pattern of parallel coalignment with the underlying collagen. They appeared to repolymerize via two different pathways: (1) a radial growth outwards from the microtubule organizing center followed by a progressive realignment with the underlying collagen and (2) a gradual and simultaneous polymerization along cold-stable, antitubulin staining fibers. These fibers were also aligned with the collagen fibrils and may be related to the aligned intermediate filaments.     

Fine Structure of the Developing Scale in the Cichlid Hemichromis bimaculatus (Pisces,Teleostei, Perciformes)

The study of the formation and structure of the early teleost scale and its associated cells has been carried out on Hemichromis bimaculatus fry using in toto staining with alizarin and transmission electron microscopy techniques. Results of the study show very rapid scale formation in Hemichromis. The papilla of the scale differentiates a little in advance of the bone scale formation. No epidermal cells are involved in the constitution of the scale pocket made up of scleroblasts. In Hemichromis, as in other teleost scales, the osseous layer is the first one to be secreted by, presumably, only the scleroblasts. Then the scleroblasts specialize in their functions. Superficial ones are involved in the formation of osseous circuli; marginal scleroblasts are responsible for growth in diameter of the scale; while deep scleroblasts allow the scales to thicken owing to the progressive addition of collagen fibrils organized in a “plywood-like” structure which constitutes the fibrillary plate of the scale. Mineralization occurs very rapidly within the osseous layer in the form of hydroxyapatite-like crystal deposits. The fibrillary plate is not yet mineralized in Hemichromis at the stages studied here, but presumably is later. Results obtained in Hemichromis are discussed against similar data available in the literature on teleost scale formation.     

Alpha-actinin is absent from the terminal segments of myofibrils and from subsarcolemmal densities in frog skeletal muscle

The presence and distribution of alpha-actinin, an actin-bundling protein, was investigated at sites where frog skeletal muscle forms junctions with tendon collagen fibers. These sites, called myotendinous junctions, are regions where myofibrils terminate and where the force of muscular contraction is transmitted from muscle cells to the substratum. An antibody manufactured to chicken smooth muscle alpha-actinin was used as a probe for alpha-actinin localization in this study. The cross-reactivity of this antibody with frog skeletal muscle alpha-actinin is demonstrated in immunoblots of one-dimensional (1D) electrophoretic separations of muscle proteins. Immunofluorescent localization of anti-alpha-actinin and electron microscopic immunolabelling confirms that the antibody binds to Z-discs with high affinity. However, in sections treated for electron microscopy with affinity-purified anti-alpha-actinin and a ferritin-conjugated, second antibody, there was no significant difference between experimental or control preparations in the number of ferritin grains overlying dense, subsarcolemmal material at junctional or non-junctional regions. Furthermore, Z-discs near myotendinous junctions displayed less binding of anti-alpha-actinin than Z-discs located several micrometers or more from the cells' termini. These findings indicate that thin filaments are not bundled by alpha-actinin near the sarcolemma. The results also provide evidence for molecular heterogeneity between Z-discs at the ends of muscle cells compared with other regions of the cell in that the terminal Z-discs of myofibrils contain very little or no alpha-actinin relative to non-terminal Z-discs.     

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.     

Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining

We have studied the distribution of myosin and tubulin molecules inside the same tissue culture cells by using two antibodies labeled with contrasting fluorochromes. Antimyosin raised against human platelet myosin was labeled with rhodamine. Antitubulin raised against sea urchin vinblastine-induced tubulin crystals was labeled with fluorescein. The two antibodies stained entirely different structures inside the same flat interphase cell: antimyosin bound to stress fibers and antitubulin bound to thin, wavy fibers thought to be individual microtubules. Compact interphase cells stained diffusely with both antibodies. From prophase through early anaphase both antibodies stained the mitotic spindle, although the fluorescence contrast between the spindle and the cytoplasm was much higher with antitubulin than with antimyosin. From anaphase through telophase, strong antimyosin staining occurred in the cleavage furrow, while antitubulin stained the region between the separated chromosomes. This study established the feasibility of high-resolution fluorescent antibody localization of pairs of motility proteins in the cytoplasm of single cells, an approach which will make it possible to map out the sites of the various contractile protein interactions in situ.     

Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method

Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.     

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.     

Ultrastructural data on the melanophores associated with the cellular elasmoid scales in Leporinus friderici (Teleostei: Ostariophysi,Anastomidae): Their putative participation in scale matrix formation

Transmission electron microscopic (TEM) examination of cellular scales of Leporinus friderici reveals the presence of melanophores associated with the hyposquama, a continuous cellular layer lining the inner surface of the scale. Hyposquamal scleroblasts synthesize the collagen fibrils forming the scale matrix. Some scleroblasts lining the deep surface of the scale margin become trapped within the collagenous matrix. Neighboring melanophores become inserted within the hyposquama. They contact the scale matrix and show morphological features resembling those of the adjacent hyposquamal scleroblasts with which they are connected and, like them, they appear to be involved in the production of the collagenous scale matrix. The present ultrastructural study favors the hypothesis that melanophores in vivo are like tumorous cell lines in vitro in that they maintain a degree of plasticity allowing changes in their phenotype according to environmental conditions. The close morpho-functional links between scale scleroblasts and melanophores suggest that they could be closely related lineages derived from the same basic stem cells and support the hypothesis that scale scleroblasts as well as melanophores are neural crest derivatives. © 1996 Wiley-Liss, Inc.     

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Improved fixation for immunofluorescence microscopy using light- activated 1,3,5-triazido-2,4,6-trinitrobenzene (TTB)

A new fixation method has been developed for immunofluorescent microscopy using the photosensitive compound 1,3,5-triazido-2,4,6-trinitrobenzene (TTB). Our results show that TTB-fixed cells are well preserved morphologically and that the cellular antigens are better preserved than conventionally fixed cells. By altering one condition at a time in the TTB fixation procedure and analyzing resulting fluorescent antitubulin staining patterns in mammalian tissue culture cells, an optimal procedure was developed. Cells fixed with TTB and stained with antitubulin, antiprekeratin, anti-intermediate filament, anti-alpha-actinin, anti-myosin, antiactin, or anticlathrin were compared with cells fixed by conventional methods and stained with the same antibody. The quality of immunofluorescence images of TTB fixed cells was the same as or better than that of conventionally fixed cells. The most dramatic improvement in image quality was seen when using antiprekeratin or antitubulin. In dividing cells, particularly in metaphase, fluorescent staining with antiactin and anti-alpha-actinin was relatively excluded from the spindle. Antimyosin, on the other hand, stained the spindle and surrounding area more heavily than the subcortical region. We suggest that after TTB fixation, the immunofluorescent patterns of these contractile proteins more closely reflect their relative concentrations in living cells. The exact mechanism for fixation by TTB is not yet known. However, our studies indicated that TTB fixation was not caused by the typical fast photoinduced nitrene diradical mechanism, but rather by some slower, temperature-dependent reaction of a photoactivation product of TTB with the cell.     

The molecular organization of myosin in stress fibers of cultured cells

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.     

Relation between cell activity and the distribution of cytoplasmic actin and myosin

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.     

Dynamic reorientation of cultured cells and stress fibers under mechanical stress from periodic stretching.

Cell lines derived from rat aorta and frog kidney were cultured on elastic membrane, and mechanical stress was given to the cells by stretching the membrane periodically. Cell reorientation oblique to the direction of stretching occurred as a result of the rapid withdrawal of cell periphery located along the direction of stretching and gradual extension of the cell membrane toward the direction oblique to the direction of stretching. Dynamic reorganization of stress fibers in living cells was visualized by labeling stress fibers with TRITC(3)-actin or EGFP-tagged moesin fragments with actin-binding ability. Stress fibers aligned in the direction of stretching disappeared soon after the start of stretching and then obliquely reoriented stress fibers appeared. The stretch-induced reorientation of cultured cells was suppressed by an inhibitor of stretch-activated (SA) cation channels and by a Ca(2+) chelator. However, the rearrangement of stress fibers was not affected by these agents. From these results, we suggest that Ca(2+) influx via SA channels is involved in stretch-induced cell reorientation but stress fiber rearrangement is independent of SA channels. Therefore, cell reorientation does not simply depend on the arrangement of stress fibers but may be controlled by some additional mechanism(s) which is regulated by calcium signaling.     

Phagocytosis induced by thyrotropin in cultured thyroid cells is associated with myosin light chain dephosphorylation and stress fiber disruption

The actin/myosin II cytoskeleton and its role in phagocytosis were examined in primary cultures of dog thyroid cells. Two (19 and 21 kD) phosphorylated light chains of myosin (P-MLC) were identified by two- dimensional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal fraction. Analyses of Triton-insoluble and soluble 32PO4-prelabeled protein fractions indicated that TSH (via cAMP) or TPA treatment of intact cells decreases the MLC phosphorylation state. Phosphoamino acid and tryptic peptide analyses of 32P-MLCs from basal cells showed phosphorylation primarily at threonine and serine residues; most of the [32P] appeared associated with a peptide containing sites typically phosphorylated by MLC kinase. Even in the presence of the agents which induced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct serine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the cytoskeletal fraction with [gamma-32P]ATP indicated that Ca2+, EGTA, or trifluoperazine (TFP) has little effect on the phosphorylation of MLC. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupted within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC dephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, induced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin, another event(s) mediated by A-kinase appears necessary for phagocytic activity.     

Cultured megakaryocytes: changes in the cytoskeleton after ADP-induced spreading

Megakaryocytes from guinea pig bone marrow were isolated and maintained in liquid culture and were treated with ADP, thrombin, arachidonic acid, or collagen. Megakaryocytes spread with an active ruffled membrane in response to ADP (1-100 microM), thrombin (1.0 U/ml), and arachidonic acid (50 microM) but responded to collagen surfaces only if fibronectin was added to the cultures. Spreading could be blocked completely by dibutyryl cyclic AMP (dibutyryl cAMP) or isobutylmethylxanthine at 1 mM, as well as by cytochalasin D (2 microgram/ml), but not by colchicine up to 1 mg/ml. The distribution of contractile proteins was examined by immunofluorescence. In untreated, spherical cells, staining with antimyosin, antifilamin, anti-alpha- actinin, or with fluorescein-labeled subfragment 1 (FITC-S1) was diffuse and unpatterned. With antitubulin antibody, however, microtubules were seen in a dense array throughout the unspread cells. In actively ruffling spreading cells, myosin, filamin, and actin were visualized in the region of the ruffled membrane while alpha-actinin was seen most prominently in a band located proximal to the inner part of the ruffle. In fully spread cells, actin, myosin, filamin, and alpha- actinin were seen in filaments that filled the cytoplasm. Antimyosin and anti-alpha-actinin staining of the filaments was periodic with approximately 1 micrometer center-to-center spacing. Actin, filamin, and alpha-actinin were also identified in punctate spots throughout the spread cytoplasm. Microtubules were absent from the ruffle but filled the cytoplasm of fully spread cells. Rings, 1.5-2.5 micrometer in diameter, were seen with antitubulin in 13% of the spread cells. Our results show that megakaryocytes respond to platelet agonists, but typically by spreading, rather than extending, filopodia. From the changes in localization of contractile proteins and from time-lapse cinematography, we propose a model for cell spreading.     

The histology and calcification of regenerating scales in the blackspotted topminnow, Fundulus olivaceus (Storer)

The regenerating scale and tissues comprising the scale pocket of Fundulus olivaceus were examined microscopically at specific intervals. Scale removal resulted in a thickening of the epidermis which persisted through the early stages of regeneration. This thickening was due in part to the appearance of columnar basal cells which divided producing cells that became mucous cells and squamous cells. The scale regenerated as a relatively large plate of bone which first appeared between layers of scleroblasts on the floor of the scale pocket and then grew producing circuli and radii. By the fourth day of regeneration, calcium was observed in the cytoplasm of the scleroblasts and at randomly distributed foci in the osseous portion of the scale. The osseous layer was completely calcified by 15 days of regeneration.