Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin, and villin in rat and bovine exocrine glands

Actin, myosin, and the actin-associated proteins tropomyosin, alpha-actinin, vinculin, and villin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by means of immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. The presence of villin, which has so far been demonstrated only in intestinal and kidney brush border, was further confirmed by antibody staining of blotted electrophoresis gels of whole acinar cell extracts. Fluorescently labelled phalloidin, which reacts specifically with F-actin, gave similar staining, within the cell apex to that obtained with antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web, antibodies to myosin and tropomyosin gave only a weak immunostaining (confined to the lateral cell borders) whereas antibodies to actin and alpha-actinin led to a rather strong bead-like staining along the lateral and basal cell membrane most probably marking microfilament-associated desmosomes. Anti-villin immunofluorescence was confined to the apical terminal web. It is suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca(++)-dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins.     

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.     

Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence

The distribution of actin and myosin in Dictyostelium amebae at different developmental stages was studied by improved immunofluorescence ("agar-overlay" technique). Both were localized at the cortical region of amebae in all early developmental stages. In amebae with polarized morphology, bright fluorescence with antiactin was seen in the anterior pseudopode. The cortex in the posterior end was also stained with antiactin. On the other hand, very specific crescent-shaped staining with antimyosin was seen at the posterior cortex. In cells in contact with each other, actin was concentrated at the contact region, whereas myosin was localized specifically in the cortex on the other side of the contact region. At the aggregation stage, when monopodial amebae migrate forming streams, actin staining was seen all around the cell periphery, with intense fluorescence in the anterior pseudopode. On the other hand, specific staining of myosin was seen only at the posterior cortex. The cleavage furrow of cells performing cytokinesis displayed distinct myosin staining, and this staining represented the filamentous structure aligned in parallel to the axis of constriction. These findings indicate that myosin staining reflects the portion of the cell cortex where contraction occurs and the motive force of ameboid movement is generated at the posterior cortex of a migrating cell.     

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.     

Interaction of contractile proteins with DNA

The interaction of contractile proteins (myosin, actin, tropomyosin and troponin) with DNA was studied in vitro using a nitrocellulose filter binding technique. The data indicate a high affinity of myosin and troponin for DNA, a lesser interaction between DNA and tropomyosin and the absence of binding of actin to DNA. When binding to DNA was detected, the interaction was higher with single-stranded DNA than with RNA or double-stranded DNA, although in some conditions myosin binds equally as well to native as to denatured eukaryotic DNA. Myosin binds better to eukaryotic than to phage native DNA.     

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.     

Polysome structure in sea urchin eggs and embryos: an electron microscopic analysis

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.     

Formation of the tooth enamel rod pattern and the cytoskeletal organization in secretory ameloblasts of the rat incisor

The localization of actin, myosin, tropomyosin, alpha-actinin, vinculin, and desmoplakin I/II was visualized by immunofluorescence microscopy. Antibodies against myosin, tropomyosin, and alpha-actinin and rhodamine-phalloidin labeled strongly the proximal and distal terminal webs which ultrastructurally consist of dense microfilament bundles. In the distal terminal web, the staining by these reagents occurred mostly perpendicular to the long axis of the incisor. Antivinculin stained the general area where the distal terminal web is located in the ameloblast. Anti-desmoplakin I/II labeled the junctional area associated with the proximal and distal terminal webs. The anti-desmoplakin staining was stronger along the cell border perpendicular to the long axis of the incisor. Comparison of the rhodamine-phalloidin staining pattern of the distal terminal web and the enamel secretion pattern by ameloblasts revealed that a change in the distal terminal web staining pattern preceded a change in the secretion pattern. These observations suggest that the cytoskeletal organization in the ameloblast is involved in the formation of the enamel matrix pattern in the rat incisor.     

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear

Summary The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.     

Human smooth muscle cell cultures of the stomach morphologic and biochemical studies

Summary Smooth muscle cell cultures were prepared from stomach explants obtained surgically from 10 patients with duodenal ulcer. The cultured cells grew in either overlapping layers in “hills and valleys” or in parallel arrays. The ultrastructure studies showed plasmalemmal vesicles, bundles of myofilaments associated with dense bodies, and gap junctions. The synthesis of contractile proteins illustrated the preponderance of actin on myosin and tropomyosin. The synthesis of contractile proteins in stomach smooth muscle cell cultures is significantly higher than in skin fibroblast cultures, i.e. 20 x higher for myosin, 10 x higher for actin, and 30 x higher for tropomyosin.     

Localization of Myosin,Actin, α-Actinin,Tropomyosin and Vinculin in Surface-Activated,Spreading Human Platelets

We observed the localization of the contractile proteins myosin, filamentous actin, α-actinin, tropomyosin, and vinculin in surface-activated, spreading human platelets using a single fluorescence staining procedure and conventional fluorescence microscopy. Myosin was distributed in a speckled pattern that extended radially from the granulomere. F-actin demonstrated cable-networks. Tropomyosin and α-actinin occurred in a punctuate distribution, and vinculin was localized at adhesion sites. Although myosin, F-actin, α-actinin, tropomyosin, and vinculin were not studied in resting platelets, our data support the idea that these contractile proteins are reorganized and reassembled in activated platelets during platelet function.     

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.     

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

Preservation of cellular polarity in isolated hepatocytes

Summary The distribution of actin, myosin and tropomyosin in freshly isolated and short-term cultured rat hepatocytes was investigated by use of both rhodaminyl-phalloidin staining and immunofluorescence techniques. The cytoskeletal proteins were mainly located in distinct areas of the hepatocyte membrane, corresponding to their accumulation in the bile-canalicular region of liver tissue. In freshly prepared cells, these sections resembled sharp, angled or branched bands, similar to the pattern of hemicanaliculi. During incubation in a monolayer culture, these bands were transformed to circular formations. Simultaneously, enclosed bile-canalicular spaces between undissociated hepatocytes were visualized by staining of actin, myosin, and tropomyosin. The preservation of canalicular cytoskeletal structures in isolated hepatocytes is an indication of cellular polarity. Our findings suggest a uniform association of membrane-bound F-actin with myosin and tropomyosin.     

Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis

Actin-binding protein (ABP) and myosin are proteins that influence the rigidity and movement, respectively, of actin filaments in vitro. We examined the distribution of ABP and myosin molecules in acetone-fixed rabbit lung macrophages by means of immunofluorescence. The staining for both of these proteins in unspread cells was quite uniform, but was reduced in the nucleus and concentrated slightly in the periphery. The peripheral accumulation of staining attenuated in uniformly spread cells, although filopodia and hyaline veils definitely stained. In cells fixed during ingestion of yeast particles, the brightest staining correlated with the disposition of organelle-excluding pseudopodia initially surrounding the yeast. After phagocytosis was complete and the yeasts resided in intracellular vacuoles, no concentration of staining around the ingested yeasts was detectable. We conclude that ABP and myosin molecules are components of the structural unit of the cell responsible for spreading and phagocytosis, the hyaline cortex, a region known to be rich in actin filaments. The findings are consistent with the theory that these molecules control the rigidity and movement of filaments in the periphery of the living macrophage.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.     

Cytoskeletal organization in locomoting cells of the V2 rabbit carcinoma

The relationship between the organization of cytoskeletal elements and locomotory activity was studied in single cells of the V2 rabbit carcinoma. Like migratory fibroblasts, and unlike colony-forming epithelial cells, these cells show a pronounced horizontal polarization, and develop a large lamella at their leading front. With affinity-purified antibodies and a combination of light and electron microscopic techniques, actin and alpha-actinin (but not myosin and tropomyosin) were found highly concentrated within the marginal region of the leading lamella, both in ruffles and in the underlying zone of contacts with the substratum. Close contacts prevailed in the locomotory cells and small focal contacts developed only in cells detaching from others. Focal contacts always contained small microfilament bundles. Reorganization of actin filaments is suggested as the fundamental event for the dynamic contact formation of the leading lamella. Large microfilament bundles (stress fibers) were absent in all stages of locomotion.Since locomotory behavior and shape changes of V2 cells are the same on glass as on the surface of a natural membrane, the rabbit mesentery, organization and distribution of contractile elements of cultured V2 cells probably reflect the in vivo situation.     

Effects of tropomyosin internal deletions on thin filament function.

Striated muscle tropomyosin spans seven actin monomers and contains seven quasi-repeating regions with loose sequence similarity. Each region contains a hypothesized actin binding motif. To examine the functions of these regions, full-length tropomyosin was compared with tropomyosin internal deletion mutants spanning either five or four actins. Actin-troponin-tropomyosin filaments lacking tropomyosin regions 2-3 exhibited calcium-sensitive regulation in in vitro motility and myosin S1 ATP hydrolysis experiments, similar to filaments with full-length tropomyosin. In contrast, filaments lacking tropomyosin regions 3-4 were inhibitory to these myosin functions. Deletion of regions 2-4, 3-5, or 4-6 had little effect on tropomyosin binding to actin in the presence of troponin or troponin-Ca(2+), or in the absence of troponin. However, all of these mutants inhibited myosin cycling. Deletion of the quasi-repeating regions diminished the prominent effect of myosin S1 on tropomyosin-actin binding. Interruption of this cooperative, myosin-tropomyosin interaction was least severe for the mutant lacking regions 2-3 and therefore correlated with inhibition of myosin cycling. Regions 3, 4, and 5 each contributed about 1.5 kcal/mol to this process, whereas regions 2 and 6 contributed much less. We suggest that a myosin-induced conformational change in actin facilitates the azimuthal repositioning of tropomyosin which is an essential part of regulation.     

Giardia spp.: Distribution of contractile proteins in the attachment organelle

Giardia spp. trophozoites isolated from rat small intestine were examined by light microscopy, electron microscopy, SDS-gel electrophoresis, and immunocytochemistry. In SDS-gels of protein extracts of isolated Giardia spp. trophozoites protein bands corresponding to myosin, α-actinin, and actin were identified by comigration with avian myofibril proteins and molecular weight standards. Actin was specifically identified in SDS-gels by immunoautoradiography. Immunostaining for actin, α-actinin, myosin, and tropomyosin in trophozoites was demonstrated in the periphery of the ventral disc in an area corresponding to the lateral crest. Electron-dense fibrillar was observed in the lateral crest of the ventral disc by electron microscopy. Immunostaining for actin and α-actinin was also observed in the area of the median body, a microtubular organelle, and in electron-dense fibrillar material associated with the intracellular axonemes of the posterior-lateral flagella. The localization of these contractile proteins in the ventral disc suggests that they may play an important role in the mechanism of trophozoite attachment.