Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.     

Rod-like elements from actin-containing microfilament bundles observed in cultured cells after treatment with cytochalasin A (CA)

Immunofluorescence microscopy using antibody against actin has been used to study the expression of microfilamentous material in cells of a cloned mouse 3T3 line during cytochalasin A (CA) induced cell contraction. A conspicuous modification of the structure of the microfilament bundles is observed. Actin containing rod-like elements can be visualized both by phase contrast and immunofluorescence microscopy. These actin containing rods are of rather defined length (approximate length 5 μm) and seem to be derived as subunits from the original microfilament bundles. In some cells the rods were in the same orientation as the microfilament bundles in control cells, whereas other cells showed scattered arrangements. The phenomenon suggests intrafibrillar periodical heterogeneity in the microfilament bundles.     

Mechanisms of cellular adhesion : II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells

cAMP/theophylline exaggerates cell shape—whether the fibroblastic morphology of controls or the epithelioid shape of colchicine-treated cells. The ultrastructural basis is that cAMP/theophylline increases the number and linearity of microtubules and microfilament bundles, although where also treated with colchicine, the cells adopt a well-spread shape maintained by microfilament bundles alone. Since interference reflection microscopy shows that colchicine promotes the marked alignment of focal contacts (which terminate microfilament bundles) it is concluded that microtubules encourage angular cell form and modify the pattern of adhesions by influencing the directionality of microfilament bundle formation although they are inessential for the maintenance of the spread form or adhesion per se.     

Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts

The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.     

Deep-etch visualization of the Sertoli cell (blood-testis) barrier in the boar.

The Sertoli cell (blood-testis) barrier in the boar was visualized by the freeze-fracture, deep-etch, rotary-replication technique. Three kinds of cross-bridging structures were clearly recognized in the following three ectoplasmic specialization (ES) regions; (1) cross-bridges in the intercellular space between adjacent Sertoli cell membranes; (2) cross-bridges in the space between the Sertoli cell membrane and microfilament bundles; and (3) cross-bridges in the space between microfilament bundles and subsurface cisternae. Results from immunolocalization, vinculin and alpha-actinin were recognized in the Sertoli cell barrier. Our findings show that these structural elements of the Sertoli cell barrier are held together by these cross-bridging structures, and provide important morphological evidence that implicates the ES in the dynamic function of the microfilament bundles of the Sertoli cell barrier.     

Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism

Immunofluorescence with an antiactin antibody and electron microscopy were used to study the distribution of actin in cultured mouse fibroblasts during treatment with inhibitors of energy metabolism. The inhibitors induce gradual disorganization of actin-containing microfilament bundles. At the first stage of the process the bundles degrade into separate fragments; later only small patches of actin can be found in the inhibitor-treated cells. This transformation takes about 90 min and is fully reversible as microfilament bundles are recovered after incubation of the cells in the inhibitor-free growth medium. The inhibitors do not alter actin distribution in the presence of glucose. This shows that their action is due to a reduction of the ATP level in the cells. A 90 min incubation with the inhibitors does not markedly alter either the cell shape or the microtubule system. Inhibitors of the energy metabolism prevent cytochalasin action on cells. Cytochalasin B (CB) or cytochalasin D (CD) rapidly disorganize the microfilament bundles and cause cell arborization. However, microfilament bundle destruction in the cells incubated in the mixture of cytochalasin and any of the inhibitors requires 90 min and is not accompanied by dramatic changes in the cell morphology, so the process is indistinguishable from microfilament bundle destruction in the presence of the inhibitors alone.     

Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy.

A procedure is described which allows the examination of the cytoskeleton of a single PtK2 cell first by immunofluorescence and then by electron microscopy after staining with uranyl acetate. The immunofluorescent patterns of these detergent resistant cytoskeletons elicited with various monospecific antibodies closely resemble the patterns found in whole cells. Comparison of the immunofluorescence and electron micrographs directly supports the previous assignments of actin, myosin, filamin, α-actinin and tropomyosin as proteins associated with microfilament bundles in non-muscle cells. Actin is also found associated with a fine lattice-like structure present both in the ruffles and lying above the microfilament bundles in the cell body. The tonofilament bundles present in PtK2 cytoskeletons are not decorated by antibodies directed against the proteins associated with microfilament bundles. Antibodies directed against tonofilaments decorate specifically this system and not the microfilament bundles.     

Microfilament organization and distribution in freeze substituted tobacco plant tissues

Summary The organization and distribution of microfilaments in freze substituted leaf tissues and root tips of tobacco plants (Nicotiana tabacum L. var. Maryland Mammoth) were investigated in detail. Three categories of microfilaments were recognized in interphase cells of all tissues including those in the root cap: (1) microfilament bundles; (2) single microfilaments; (3) cortical microtubuleassociated microfilaments. While the microfilament bundles appeared to be distributed throughout the cytoplasm, the single microfilaments were mainly confined to the cell periphery. All three categories of microfilaments were associated with various organelles. Our study indicates that the three categories of microfilaments are normal cytoskeletal components in higher plant cells. The implications of these findings are discussed.Abbreviations MFB microfilament bundle - SMF single microfilament - MAMF microtubule-associated microfilament - AAP actin-associated protein - MAP microtubule-associated protein - MES 2(N-morpholino)ethanesulfonic acid     

Distribution of microfilament bundles during rotation of the nucleus in 3T3 cells treated with monensin

Cytoskeletal aspects of monensin-treated 3T3 cells with rotating nuclei were studied by immunofluorescence. The pattern of intermediate filaments and microtubules appeared unchanged when compared with control cells having a stationary nucleus. In contrast, the actin microfilament bundles appeared to have a consistent distribution in cells with rotating nuclei. Typically, we did not find long microfilament bundles that traverse the length of the cytoplasm of cells that were fixed at the time of nuclear rotation. Instead, there was a local distribution of short microfilament bundles situated ventrally to the nucleus and oriented at various angles to one another and to the predominant distribution of microfilament bundles in the cell. The observations suggest that the actin cytoskeleton is reorganized locally before or during rotation of the nucleus.     

Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.     

Regulating role of the microtubule system in the distribution of actin microfilament bundles in embryonic fibroblasts

The effect of colcemid on the distribution of actin microfilament bundles in the mouse embryo fibroblasts was studied using immunomorphological methods. In the control fibroblasts, microfilament bundles usually cross the entire cell and are oriented in parallel to the stable edges of the cell. In the colcemid-treated cells there are several groups of bundles. In each group all bundles are oriented in the same direction but these directions do not depend on the cell shape. Besides, bundles in the colcemid-treated cells are shorter than in the control cells. Microtubules are suggested to control the organization of action bundles.     

Microfilament bundles and the control of pinocytotic vesicle distribution at the surfaces of normal and transformed fibroblasts

The surfaces of human WI38 and hamster embryo fibroblasts, and those of their SV40-transformed derivatives (WI38-VA13, wt/HEF, and ts A28/HEF), were freeze-etched and thin-sectioned in situ, to study the effects of transformation-related changes in microfilament bundles on plasma membrane architecture. Quantitative electron microscopic analysis showed that pinocytotic vesicles on the non-transformed cell surface frequently (48–63% of the samples) were arranged in ordered linear or fusiform aggregates delineated by longitudinal surface ridges and furrows. These longitudinal structures appeared to result from interactions between microfilament bundles and the plasmalemma. Transformed cell surfaces did not have many longitudinal microfilament-bundle impressions, and most of their pinocytotic vesicle aggregates (70–80%) were irregular in shape and composed of randomly scattered vesicles. Microfilament bundles present in both types of transformants were reduced significantly in thickness (p < 0.001). These changes were temperature sensitive in the ts A28/HEF system. The data strongly suggest that the distribution of pinocytotic vesicles at the fibroblast surface is influenced by the disposition of cortical microfilament bundles, and that this control mechanism is disrupted during cellular transformation.     

Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels

Myosin, tropomyosin, and actin were localized in the epithelial cells of rat intestine by means of specific antibodies to chicken gizzard smooth muscle myosin, tropomyosin, and actin by immunohistochemical studies at both the light and electron microscope levels (unlabeled antibody enzyme technique). The pattern of antibody staining was the following (a) Anti-actin was associated with the microfilament bundles of the microvilli in their entire length, as well as with the microfilament network in the terminal web. (b) Anti-myosin was concentrated along the rootlets of the microvillar microfilament bundles and within the filamentous feltwork forming the terminal web. (c) Anti-tropomyosin showed a distribution similar to that of anti- myosin. In addition, the three antibodies also labeled the subplasmalemmal web underneath the cell membrane bordering on the basal lamina. Utilizing the above ultrastructural findings, we wish to propose a functional model of microvillar contraction.     

Role of fibronectin in the organisation of the cytoskeleton during the spreading of rat mammary epithelial cells.

The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor.     

Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.     

Association of microfilament bundles with lysosomes in polymorphonuclear leukocytes

The juxtaposition of microfilament bundles and lysosomes seen both in thin-sectioned cells in the transmission electron microscope and in cryofractured cells in the scanning electron microscope, and the presence of short filamentous structures between lysosomes and microfilament bundles, suggest that microfilaments may be attached to lysosomal membranes and that these filaments may be involved in lysosomal movements. Further work is in progress to test these hypotheses.     

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg²⁺ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.