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.     

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.     

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.     

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.     

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.     

The microfilament pattern in the somatic follicle cells of mid-vitellogenic ovarian follicles of Drosophila

The microfilament pattern in the somatic follicle cells of mid-vitellogenic stage 9 to 11 follicles of Drosophila was analyzed by staining F-actin with fluorescence-labeled phalloidin. During the analyzed stages of oogenesis, the follicular epithelium differentiates morphologically and functionally. These changes are also reflected at the organization of the microfilaments. At stage 10, they show no preferred orientation in the very thin follicle cells covering the nurse cells. In contrast, the microfilaments in the basal part of the columnar follicle cells covering the oocyte become organized in parallel bundles oriented perpendicular to the long axis of the follicle. During stages 10B/11 this organization is maintained at the nurse cell/oocyte border but becomes more sloppy towards the posterior pole of the follicle. The basal part of the follicle cells containing the microfilament bundles adheres so tightly to the basement membrane that this acellular layer cannot be separated mechanically from the epithelium. Indirect evidence from inhibition studies with cytochalasins and the effects of collagenase or pronase E added to the culture medium suggest that the microfilament bundles may promote increased adhesiveness of the follicle cells to the basement membrane. The possible functional implications of the microfilaments and their orientation are discussed.     

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.     

Morphological evidence for cyclic AMP-induced reverse transformation in vole cells infected with avian sarcoma virus.

Normal fibroblasts of the vole displayed moderately spread or flattened, spindle-shaped, or polygonal morphologies and attached firmly to a substrate. Topographic features of these cells included sparse microvilli, ruffles, and filopodia. Microfilament bundles, intermediate filaments, and long microtubules generally parallel to each other, and the long axis of the cell or its extensions were present in the cytoplasm. Fibronectin was abundant, and fibronectin fibrils often formed junctions at the cell membrane with microfilament bundles. Transformation with avian sarcoma virus converted 90% of the cells to spheres 5 to 10 microns in diameter. In contrast to the normal vole cells, microfilament bundles were absent, microtubules were short and randomly arranged, and fibronectin was no longer visible. Exposure to dibutyryl cyclic AMP and testololactone caused a majority of the spherical cells to stretch and flatten, a process referred to as reverse transformation. Microtubules radiated out to the cell periphery and became parallel in cell extensions, while long microfilament bundles appeared in the cytoplasm. Parallel intermediate filaments were arranged throughout the cell. This ultrastructural analysis of reverse transformation in avian sarcoma virus-transformed vole cells detailed the status of the cytoskeletal system and showed agreement with earlier findings (Puck et al., J. Cell. Physiol. 107:399-412, 1981) using indirect immunofluorescence.     

Extracellular and Intracellular Correlates of Organ Initiation in the Embryonic Chick Thyroid

Apical and basal bundles of microfilaments have been suggestedas intracellular structures which might cause cellular contractionsleading to bending movements during organogenesis. Microfilamentbundles may have a different role in some organ primordia. Theembryonic chick thyroid develops as an evagination of the pharyngealfloor at the level of the anterior attachment of the heart.The developing thyroid is confined to a pocket surrounded bythe developing aortic arches, ventral aorta, and truncus arteriosus.Initiation of thyroid evagination is accompanied by the formationof grooves as two concentric rings in the basal surface of theplacode near its margins. The cells surrounding the groovescontain longitudinally oriented bundles of microfilaments andmicrotubules. Other microfilament bundles are present at thecell apices and bases. The primordium almost doubles in volumeduring the period of evagination, but the area of pharyngealfloor that it occupies undergoes virtually no increase. A modelis presented which describes the role of microfilament bundlesduring thyroid evagination as stabilizing cellular dimensions.The bending movements are thought to result from cell elongation,increase in cell numbers, and confinement of the thyroid toa limited space by the developing truncus arteriosus and itsbranches.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

A seasonal cycle of cell wall structure is accompanied by a cyclical rearrangement of cortical microtubules in fusiform cambial cells within taproots of Aesculus hippocastanum (Hippocastanaceae)

Aspects of the structure and ultrastructure of the fusiform cambial cells of the taproot of Aesculus hippocastanum L. (horse chestnut) are described in relation to the seasonal cycle of cambial activity and dormancy. Particular attention is directed at cell walls and the microtubule and microfilament components of the cytoskeleton, using a range of cytochemical and immunolocalization techniques at the optical and electron-microscopical levels. During the dormant phase, cambial cell walls are thick and multi-layered, the cells possess a helical array of cortical microtubules, and microfilament bundles are oriented axially. In the early stages of reactivation, vesicle-like profiles are associated with the cell walls, whereas arrangement of the cytoskeletal elements remains unchanged. In the succeeding active phase, the cell walls are thin, and cortical microtubules form a random array, although microfilament bundles maintain a near-axial orientation. The observations are discussed in relation to the seasonal cycle of wall structure and cortical microtubule rearrangement within the vascular cambium of hardwood trees. It is suggested that the cell-wall thickening at the onset of cambial dormancy, which is associated with the presence of a helical cortical microtubule array, should be considered to be secondary wall thickening, and that selective lysis of this secondary wall layer during cambial reactivation restores the thinner, primary wall found around active cambial cells.     

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.     

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.     

A primary role for microfilaments, but not microtubules, in hormone-induced cytoplasmic retraction

The distributions of microfilaments and microtubules were studied during transient hormone-induced changes in cell shape (retraction-respreading). Two cell types (fibroblasts and bone cells), differentially responsive to parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂), were analysed. The cytoplasm of fibroblasts retracted in response to PGE₂ but not PTH, whereas bone cells could respond to both PGE₂ and PTH. Time-lapse photomicrography indicated that the retraction began within minutes of hormone addition, while respreading occurred over longer times, up to 8 h. Affinity-purified actin and tubulin antibodies were used to follow the appearance of microtubules and microfilaments during both the retraction and the respreading phases. Microtubules appeared not to reorganize noticeably, although they were squeezed closer together in cellular pseudopods; no extensive loss or growth was detectable. Microfilaments did alter drastically their appearance and distributions. Soon after hormone addition when earliest detectable cytoplasmic retraction was evident, microfilament bundles appeared to break down. Remaining microfilament bundles consisted of relatively short, non-aligned fragments or aggregates. During respreading, microfilament bundles regrew and realigned throughout the cytoplasm. These data suggest a primary role for microfilaments, but probably not microtubules, in these cell shape changes.     

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.     

Stages in specialization of fibroblast adhesion and deposition of extracellular matrix

Fibroblast cells seeded on a serum glycoprotein shown previously to mediate a spread shape without focal adhesions or microfilament bundles (Stage 1 spread) are now shown to have substratum contacts in which coated pits are abundant and associated with small globular deposits of glycocalyx bridging to substratum and staining for fibronectin and acidic glycoconjugates. After stimulation with serum or fibronectin to form focal adhesions and microfilament bundles (Stage 2 spread), clathrin-based structures remain at the cell underside but no longer in conspicuously higher concentration than on the dorsal surface; extracellular material at adhesions is now as regular strands which stain for acidic glycoconjugates but (as reported earlier by Chen and Singer) not always for fibronectin. During these stages of adhesion, striking changes are seen in the cellular display of fibronectin monitored by immunofluorescence. In rounded cells this is granular and cytoplasmic, concentrated around the submembranous cortex; on spreading to Stage 1, it remains granular and intracellular but is now oriented strongly towards the lower cell surface; only in Stage 2 does externalisation proceed to deposit fibrillar fibronectin on the substratum. While cytoplasmic orientation of matrix precursors can be determined by cell contact, organised externalisation is therefore coupled to fully developed adhesion status.     

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.     

Response of C3H/10T1/2 fibroblasts to an external steady electric field stimulation. Reorientation, shape change, ConA receptor and intramembranous particle distribution and cytoskeleton reorganization

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 15 V/cm. The percentage of spindle-shaped cells increased with the field strength and duration of the stimulation. These cells oriented preferentially with their long axis perpendicular to the field direction. A small percentage of the cells were found to move slightly toward the cathode during the course of electric stimulation. Although no apparent field-induced redistribution of fluorescent-labelled concanavalin A (conA) receptor along the cell periphery was observed, the bright perinuclear area appeared preferentially on the anode side. Correlative fluorescence and scanning electron microscopy (SEM) revealed no difference in the density of conA-gold microsphere labels on either side of the cell. The density of intramembranous particles on the E-face of the plasma membrane was 54% higher on the anode side than on the cathode side of the cell. The microfilament bundles were observed to be disrupted after 30 min of 10 V/cm stimulation by rhodamine phalloidin labelling of F-actin. The cell sensitivity to electric field-induced reorientation and cell shape changes was reduced by pretreatment with conA, and to a lesser extent, with succinyl conA or wheat germ agglutinin (WGA). ConA pretreatment alone also reduced the prominence of microfilament bundles. However, post-field lectin binding to the cell has no effect on cell recovery. It is possible that the generally flat 10T1/2 cells retract and realign in order to minimize the disruption of their membrane potential. The conA binding-mediated receptor-cytoskeletal linkage temporarily immobilizes the cell and inhibits subsequent field-induced shape changes.     

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.     

Normal and transformed fibroblast spreading: Role of microfilament polymerization and actin-myosin contractility

The polymerization of microfilaments and their subsequent rearrangements under the control of actin-myosin interactions are two major processes that underlie the morphogenetic reactions of cells. We studied their role in the spreading of normal and transformed REF52tetRas fibroblasts with adjustable ras-oncogene expression. Treatment with inhibitors of cell contractility (Y27632 or blebbistatin) led to the disappearance of actin bundles and focal adhesions; however, pseudopodial activity in both normal and transformed cells remained high. Under these conditions, spreading was more accelerated in normal cells then in ras-transformed cells. In normal cells treated with low concentrations of latrunculin A actin polymerization was suppressed, stress fibers and focal adhesions were preserved, but lamellipodial activity was lost and spreading was dramatically inhibited. In transformed fibroblasts treated with low doses of latrunculin, actin bundles and focal adhesions almost disappeared, but pseudopodial activity was apparent and spreading was less suppressed. Therefore, the most significant process in the regulation of cell spreading and polarization is the microfilament polymerization at the leading edge. ras-Transformed cells are less sensitive to inhibitors that affecting the cytoskeletal structure than nontransformed cells. Possible mechanisms that underlie the difference are discussed.