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.     

Phorbol myristate acetate stimulates microtubule and 10-nm filament extension and lysosome redistribution in mouse macrophages

Phorbol myristate acetate (PMA) stimulates cell spreading and fluid- phase pinocytosis in mouse peritoneal macrophages. Colchicine (10(-5) M) and cytochalasin B (10(-5) M) abolish PMA stimulated pinocytosis but have little effect on cellular spreading (Phaire-Washington et al., 1980, J. Cell Biol., 86:634-640). We report here that PMA also alters the organization of the cytoskeleton and the distrubution of organelles in these cells. Neither control nor PMA-treated macrophages contain actin cables. PMA-treated resident thioglycolate-elicited macrophages exhibit beneath their substrate-adherent membranes many randomly distributed punctate foci that stain brightly for actin. The appearance and distribution of these actin-containing foci are not altered by colchicine (10(-5) M) or cytochalasin B (10(-5) M). In thioglycolate- elicited macrophages PMA causes the extension and radial organization of microtubules and 10-nm filaments and promotes the movement of secondary lysosomes from their perinuclear location to the peripheral cytoplasm. Depending upon the concentration of PMA used, 45-71% of thioglycolate-elicited macrophages and 32-44% of proteose-peptone- elicited macrophages and numerous lysosomes, radiating from the centrosphere region, arranged linearly along microtubule and 10-nm filament bundles. Colchicine (10(-5) M) and podophyllotoxin (10(-5) M) prevent the radial redistribution of microtubules, 10-nm filaments, and lysosomes in these cells. Cytochalasins B and D (10(-5) M) have no inhibitory effects on these processes. These findings indicate that microtubules and 10-nm filaments respond in a coordinated fashion to PMA and to agents that inhibit microtubule function; they suggest that these cytoskeletal elements regulate the movement and distribution of lysosomes in the macrophage cytoplasm.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Structural Aspects of Saltatory Particle Movement

A variety of cells possess particles which show movements statistically different from Brownian movements. They are characterized by discontinuous jumps of 2–30 µ at velocities of 0.5–5 µ/sec or more. A detailed analysis of these saltatory, jumplike movements makes it most likely that they are caused by transmission of force to the particles by a fiber system in the cell outside of the particle itself. Work with isolated droplets of cytoplasm from algae demonstrates a set of fibers involved in both cytoplasmic streaming and saltatory motion, suggesting that both phenomena are related to the same force-generating set of fibers. Analysis of a variety of systems in which streaming and/or saltatory movement occurs reveals two types of fiber systems spatially correlated with the movement, microtubules and 50 A microfilaments. The fibers in Nitella (alga) are of the microfilament type. In other systems (melanocyte processes, mitotic apparatus, nerve axons) microtubules occur. A suggestion is made, based on work on cilia, that a microtubule-microfilament complex may be present in those cases in which only microtubules appear to be present, with the microfilament closely associated with or buried in the microtubule wall. If so, then microfilaments, structurally similar to smooth muscle filaments, may be a force-generating element in a very wide variety of saltatory and streaming phenomena.     

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.     

10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin

Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha- or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments. The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha- and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha- and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells. The filament collapsing was observed to be complete at 1.5-2 h after injection. During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region. By 1.5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed. Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly. Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninjected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism. These results suggest that through a direct interaction of antibodies with either alpha- or beta- tubulin subunits, 10-nm filaments were dissociated from their normal distribution. It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

Effects of injected inhibitors of microfilament and microtubule function on the gastrulation movement in Xenopus laevis

It was suggested recently that gastrulation movements in amphibian embryos are caused by the active cell locomotion of individual cells. In order to elucidate the role of microfilaments and microtubules in the cell locomotion occurring during gastrulation, cytochalasin B, colchicine, and other microtubule-disrupting drugs were injected into the blastocoel of early gastrulae of Xenopus laevis. Hypertonic solutions of sorbitol were also injected to elucidate the influence of the internal hydrostatic pressure on the migrating cells. The effects were examined in 1-μm Epon sections of serially fixed embryos and by transmission electron microscopy. Cytochalasin B strongly inhibits cell migration even under conditions that do not cause dissociation into single cells. The cells become round, and have only a few thin cell processes. Electron microscopy shows an alteration in the cortical microfilament network. Colchicine and other microtubule-disrupting drugs have little effect on the rate of cell migration before they cause the accumulation of many mitotic cells and the dissociation of the embryo. The interphase cells are angular and have thin processes like those in the control embryos. The microtubules disappear, and bundles of 10-nm filaments are observed in the cytoplasm of colchicine-injected embryos. Hypertonic sorbitol solutions strongly inhibit cell migration.     

Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles

The three-dimensional organization and fine structure of cytoplasmic components within whole non-embedded bovine aortic endothelial cells were examined during their attachment and spreading in tissue culture. Cells were cultured directly on Formvar-coated gold grids, fixed in glutaraldehyde and osmium tetroxide, critical point dried and examined by transmission electron microscopy (TEM) using stereoscopic methods, and by scanning electron microscopy (SEM). Reorganization of cytoplasmic structures during cell spreading occurred in four sequential stages: (1) spreading of the plasma membrane and unstructured cytoplasmic matrix; (2) spreading of cytoplasmic fiber systems (microtubules, microfilament bundles and microtrabecular system); (3) alignment of microfilament bundles and formation of radial tracts of microtubules; and (4) centripetal movement of organelles along radial tracts. These stages observed by TEM correlated with progressive degrees of cell flattening as visualized by SEM. These studies demonstrate that a characteristic reorganization of intracellular fiber systems and organelles accompanies the spreading of endothelial cells in culture.     

Colocalisation of acetylated microtubules, glial filaments, and mitochondria in astrocytes in vitro

We have recently shown that acetylated alpha-tubulin containing microtubules (acetyl-MTs; labeled by antibody 6-11B-1) constitute a cold-stable subset of the microtubule network of nonneuronal cells in rat primary forebrain cultures [Cambray-Deakin and Burgoyne: Cell Motil. 8(3):284-291, 1987b]. In contrast, tyrosinated alpha-tubulin containing MTs (tyr-MTs; labeled by antibody YL1/2) are cold-labile. Here we have examined the distribution of acetyl-MTs and tyr-MTs in cultures of newborn rat forebrain astrocytes and simultaneously investigated the distribution of mitochondria and glial filaments. In double-label immunofluorescence experiments a marked colocalisation of acetyl-MTs and glial filament bundles was observed. Tyr-MTs did not show a similar colocalisation with glial filament bundles. Furthermore, the distribution of mitochondria closely followed that of the acetyl-MT and glial filament bundles. When cells were exposed to short-term (30-min) treatments with MT-disrupting agents such as colchicine and nocodazole, the tyr-MT network was removed but the distributions of acetyl-MTs, glial filaments, and mitochondria were unchanged. Increased exposure to colchicine (9-16 hr) caused a progressive disruption of the acetyl-MTs and the collapse of glial filaments and mitochondria to the perinuclear region. These results suggest that acetyl-MTs and glial filaments but not tyr-MTs may be involved in the intracellular transport of organelles and/or in the control of their cytoplasmic distribution.     

A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells

Native intermediate filament (IF) preparations from the baby hamster kidney fibroblastic cell line (BHK-21) contain a number of minor polypeptides in addition to the IF structural subunit proteins desmin, a 54,000-mol-wt protein, and vimentin, a 55,000-mol-wt protein. A monoclonal antibody was produced that reached exclusively with a high molecular weight (300,000) protein representative of these minor proteins. Immunological methods and comparative peptide mapping techniques demonstrated that the 300,000-mol-wt species was biochemically distinct from the 54,000- and 55,000-mol-wt proteins. Double-label immunofluorescence observations on spread BHK cells using this monoclonal antibody and a rabbit polyclonal antibody directed against the 54,000- and 55,000-mol-wt proteins showed that the 300,000-mol-wt species co-distributed with IF in a fibrous pattern. In cells treated with colchicine or those in the early stages of spreading, double-labeling with these antibodies revealed the co-existence of the respective antigens in the juxtanuclear cap of IF that is characteristic of cells in these physiological states. After colchicine removal, or in the late stages of cell spreading, the 300,00-mol-wt species and the IF subunits redistributed to their normal, highly coincident cytoplasmic patterns. Ultrastructural localization by the immunogold technique using the monoclonal antibody supported the light microscopic findings in that the 300,000-mol-wt species was associated with IF in the several physiological and morphological cell states investigated. The gold particle pattern was less intimately associated with IF than that defined by anti-54/55 and was one of non-uniform distribution along IF, being clustered primarily at points of proximity between IF, where an amorphous, proteinaceous material was often the labeled element. Occasionally, "bridges" of label were seen extending outward from such clusters on IF. Gold particles were infrequently bound to microtubules, microfilaments, or other cellular organelles, and when so, IF were usually contiguous. During multiple cycles of in vitro disassembly/assembly of the IF from native preparations, the 300,000-mol-wt protein remained in the fraction containing the 54,000- and 55,000-mol-wt structural subunits, whether the latter were in the soluble state or pelleted as formed filaments. In keeping with the nomenclature developed for the microtubule-associated proteins (MAPs), the acronym IFAP-300K (intermediate filament associated protein) is proposed for this molecule.     

2,5-hexanedione aggregates vimentin-, but not keratin-, intermediate filaments of PtK1 cells

2,5-hexanedione (2,5HD) induces focal accumulation of neurofilaments in nerve axons and juxtanuclear aggregation of vimentin-intermediate filaments (vimentin-IF) in cultured human skin fibroblasts. It has been postulated that 2,5HD prevents the cross-filament associations of intermediate filaments (IF) with microtubules which are required for their transport. If this is true, only subclasses of IF which depend on microtubules for their cellular distribution should be affected by 2,5HD-treatment and the aggregates formed should resemble the juxtanuclear coils which form following dissolution of microtubules by colchicine. We have tested this hypothesis in PtK1 cells which contain two separate networks of IF: vimentin-IF which aggregate in the presence of colchicine, and keratin-filaments (keratin-IF) whose distribution is not altered by depolymerization of microtubules. Treatment of confluent monolayers of PtK1 cells with 2,5HD (4 to 6 mM for 14 to 21 days) induced aggregates of vimentin-IF which resembled those induced by colchicine (5 X 10(-6)M for 48 hours), but had no effect on the distribution of keratin-IF.     

Microtubules and Microfilaments in Amphibian Neurulation

In the salamander embryo, the morphogenetic movements of neurulationare correlated with two cell shape changes in the neural epithelium:elongation and apical constriction of the columnar neural platecells. Cells first elongate to form the flat open neural plateand then constrict apically as the plate rolls up to form theneural tube. Evidence is presented that these cell shape changesare intrinsic to the cells themselves and that they play a causalrole in the morphogenetic movements. Neural plate cells containnumerous microtubules oriented parallel to the axis of elongation.These microtubules are critical to the elongation process. Possiblemechanisms for microtubule function in cell elongation are considered.During apical constriction the cells contain bundles of microfilamentswhich encircle the cell apex in purse-string fashion. Evidenceis presented which suggests that microfilament bundles playan active role in apical constriction, and that this localizedcontraction is produced by filament sliding.     

Cellular organelle transport and positioning by plasma streaming

Our analysis of known data reveals that translocations of passively movable cellular organelles from tiny granules up to large cell nuclei can be ascribed to transport by streaming cytoplasm. The various behaviours, such as velocity changes during more or less interrupted movements, forth and back shuttling and particle rotation result from different types of plasma circulation. Fast movements over long distances, as observed in the large characean internodial cells occur in strong streams generated by myosin in bundles of actin filaments in the direction of the barbed filament ends. Slow movements with frequent reversions of the direction are typical for neuronal axons, in which an anterograde plasma flow, produced in a thin layer of membrane-attached actin filaments, is compensated by a retrograde stream, produced by dynein activity in the central bundle of microtubules. Here particle rotation is due to steep flow velocity gradients, and frequent changes of particle movements result from minor particle displacements in radial directions. Similar shuttling of pigment granules in the lobes of epidermal chromatophores results from the same mechanism, whereby the centrifugal movement along astral microtubules is due to flow generated by excess of kinesin activity and the centripetal movement to the plasma recycling through the intermicrotubular space. If the streaming pattern is reversed by switching to excess dynein activity, the moving granules are trapped in the high microtubule density at the aster center. The presence of larger bodies in asters disturbs the regular, kinesin-dependent microtubule distribution in such a way that a superimposed centrifugal plasma flow develops in the microtubule-dense layer along them, which is recycled in the microtubule-free space, created by their presence. Consequently, at excess kinesin activity, nuclei, mitochondria as well as chromosome fragments move towards the aster center until they reach a dynamically stabilized position that depends on the local microtubule density. These various behaviours are not rationally explainable by models based on a mechanical stepping along microtubules or actin filaments.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

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

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

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.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.     

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.