Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.     

Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells

After trypsinization and replating, BHK-21 cells spread and change shape from a rounded to a fibroblastic form. Time-lapse movies of spreading cells reveal that organelles are redistributed by saltatory movements from a juxtanuclear position into the expanding regions of cytoplasm. Bidirectional saltations are seen along the long axes of fully spread cells. As the spreading process progresses, the pattern of saltatory movements changes and the average speed of saltations increases from 1.7 MICROMETER/S during the early stages of spreading to 2.3 micrometer/s in fully spread cells. Correlative electron microscope studies indicate that the patterns of saltatory movements that lead to the redistribution of organelles during spreading are closely related to changes in the degree of assembly, organization, and distribution of microtubules and 10-nm filaments. Colchicine (10 microgram/ml of culture medium) reversibly disassembles the microtubule-10-nm filament complexes which form during cell spreading. This treatment results in the disappearance of microtubules and the appearance of a juxtanuclear accumulation of 10-nm filaments. These changes closely parallel an inhibition of saltatory movements. Within 30 min after the addition of the colchicine, pseudopod-like extensions form rapidly at the cell periphery, and adjacent organelles are seen to stream into them. The pseudopods contain extensive arrays of actinlike microfilament bundles which bind skeletal-muscle heavy meromyosin (HMM). Therefore, in the presence of colchicine, intracellular movements are altered from a normal saltatory pattern into a pattern reminiscent of the type of cytoplasmic streaming seen in amoeboid organisms. The streaming may reflect either the activity or the contractility of submembranous microfilament bundles. Streaming activity is not seen in cells containing well-organized microtubule-10-nm filament complexes.     

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.     

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.     

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.     

Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture

Microtubules and microfilament patterns in cultured astrocytes were revealed by using indirect immunofluorescent microscopy in conjunction with anti-tubulin immune serum and anti-actin immunoglobulins respectively. In flat epitheloid astroglial cells (either polygonal or elongated) colchicine-sensitive immunofluorescent fibres, which correspond to bundles of microtubules, extend from the perinuclear cytoplasm into the cell periphery by running for long distances through the different focal planes. These patterns of organization differ markedly from the patterns of organization of microfilaments which are arranged in fibres parallel to each other and often oriented along the cell boundary. In response to the combined treatments of serum withdrawal and administration of dBcAMP, flat epitheloid astrocytes adopt a morphology similar to that of the mature astrocytes in situ in the CNS, that is of stellate process-bearing cells. This is prevented or is reverted by the administration of colchicine at the appropriate times. There are strong suggestions indicating that during cell processes formation the microtubular network is reorganized and microtubules assembled into dense bundles which are oriented along the axis of the cell processes. In view of these results, we suggest that, in contrast to microfilaments, microtubules are not determinant for the maintenance of cellular shape in elongated or polygonal flat epitheloid astroglial cells but they are required for both the formation and maintenance of processes in stellate astrocytes.     

Hormonally induced changes in the cytoskeleton of human thyroid cells in culture

Serially cultivated thyroid follicular cells are not active in hormone synthesis but retain a thyrotropin-responsive adenylate cyclase. The exposure of such cells to thyrotropin leads to an increase in the concentration of intracellular cAMP and a drastic change in morphology including a total cytoplasmic arborization. The present communication describes these changes at the cytoskeletal level using a cell line derived from a human functioning thyroid adenoma. Phase contrast microscopy showed that the cytoplasmic arborization was preceded by a total disappearance of stress fibers, visible within 20 min of exposure. Small marginal membrane ruffles could also be seen. These morphological changes could also be induced by the addition of dibutyryl cAMP. The action of both thyrotropin and dibutyryl cAMP was potentiated by theophylline. High voltage electron microscopy of whole mounted cells confirmed the loss of stress fibers (microfilament bundles). In addition, thyrotropin treatment led to an uneven redistribution of the cytoplasmic ground substance and to changes in the organization of the microtrabecular lattice. Stereo images demonstrated numerous minute surface ruffles. The thyrotropin-induced arborization was reversible even in the presence of thyrotropin. After 24 h of treatment, cells had flattened and then contained very straight and condensed microfilament bundles. The results thus demonstrate that thyrotropin induces a disintegration of microfilament bundles in human, partially dedifferentiated, follicular cells and that this effect to all appearances is caused by cAMP, the second messenger in thyrotropin action. The relation of this event in partially dedifferentiated cells to the effect of thyrotropin in the intact thyroid gland is unclear. The fact that several other cultured hormone-responsive cells round up or become arborized in conjunction with an increase in cAMP levels implies that cAMP may be a major factor in the disassembly of microfilament bundles in these cells.     

Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.     

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

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.     

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.     

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.     

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.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

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.     

Hormonally induced cell shape changes in cultured rat ovarian granulosa cells

Cultured rat ovarian granulosa cells undergo a dramatic morphological change when exposed to follicle-stimulating hormone (FSH). Exposure to FSH causes the flattened epithelioid granulosa cells to assume a nearly spherical shape while retaining cytoplasmic processes which contact the substrate as well as adjacent cells. This effect of FSH is preceded by a dose-dependent increase in intracellular cAMP, is potentiated by cyclic nucleotide phosphodiesterase inhibitors, and is mimicked by dibutyryl cAMP. Prostaglandins E1 or E2 and cholera enterotoxin also cause the cells to change shape. A subpopulation of the cells responds to luteinizing hormone. These morphological changes, which are blocked by 2,4-dinitrophenol, resemble those produced by treating cultures with cytochalasin B. Electron microscopy shows that the unstimulated, flattened cells contain bundles of microfilaments particularly in the cortical and basal regions. After FSH stimulation, microfilament bundles are not found in the rounded granulosa cell bodies but they are present in the thin cytoplasmic processes. These data suggest that the morphological change results from a cAMP-mediated, energy-dependent mechanism that may involve the alteration of microfilaments in these cells.     

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.     

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.     

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.