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.     

Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat

Antitubulin, phalloidin, and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell. A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.     

Hormonal control of morphogenetic cell death of the wing hypodermis in Lucilia cuprina.

1. Cytoplasmic fragments in the haemolymph of newly emerged flies derive from the degenerating wing hypodermis. 2. At the time of eclosion, dorsal and ventral cell layers of the wing are connected by processes containing bundles of microtubules and microfilaments. Cytoplasmic fragments contain similar bundles of microtubules but few microfilaments. 3. Extensive vacuolation marks the onset of hormonally initiated fragmentation of the wing hypodermal cells. Haemocytes containing lysosomes are present in the wing at this time, but do not invade the fragmenting hypodermis.     

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.     

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.     

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.     

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.     

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.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

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.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

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.     

Relations between cell volume control, microfilaments and microtubules networks in T2 and PC12 cultured cells

The possible relations between cell volume, microfilaments and microtubules networks have been studied in cultured mice fibrosarcoma cells of line T2 and rat pheochromocytoma cells of line PC12. The obtained results show that: 1. Changes in volume induced by application of hypo-osmotic medium are concomitant with a modification in the organization of the microfilaments network as visualized by immunocytochemistry. The microtubules lattice is not affected in these conditions. 2. Disruption of the microfilaments network by cytochalasin B causes a significant decrease in cell volume in isosmotic conditions. It also deeply affects the volume regulation response of cells swollen in hypo-osmotic media. 3. Disruption of the microtubules lattice by colchicine has no effect on volume in isosmotic conditions nor on the volume regulation that follows application of hypo-osmotic shock. The possible role of microfilaments in cell volume control is discussed.     

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.     

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.     

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.     

Patterns of actin filaments during cell shaping in developing mesophyll of wheat (Triticum aestivum L.).

Young leaves of wheat exhibit a smooth developmental gradient with meristematic cells at the base and highly differentiated cells at the tip. During differentiation, mesophyll cells attain a lobed outline resembling tube-shaped balloons with almost regularly spaced isthmi. Microfilament patterns in developing wheat mesophyll cells were investigated using fluorescent-labeled phalloidin. Various patterns were found, including delicate arrays of transversely oriented microfilaments in the cortex of the cytoplasm. A close correlation between changes in the patterns of cortical microfilaments, microtubules, cell wall microfibrils, and cell shape was observed. The fine arrays of transversely oriented microfilaments coaligned with bands of microtubules occurring during cell elongation. These bands were found beneath sites of intense wall deposition. It has recently been proposed that the resulting hoops of wall reinforcement prevent cell expansion in the corresponding regions and thus give rise to the peculiar cell shape. When cell expansion ceased, and the typical lobed cell shape was attained, a dense network of microfilaments was retained in the cytoplasm, which was in contrast to what has been described for the microtubular arrays.     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Focal contacts and the cytoskeleton

Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.     

丁酸对体外培养的人鼻咽癌上皮细胞的影响

无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞