Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

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.     

The distribution of actin in non-muscle cells : The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells

Living mouse 3T3 cells display a complex array of fibrous structures which are visible with phase contrast, Nomarski and polarized light optics. When cells are fixed and stained for indirect immunofluorescence with actin antibody, the same fibers show intense fluorescence indicating that they contain actin. Electron microscopy reveals that these fibrous structures consist of submembranous bundles of microfilaments located primarily on the attached side of the cells. The results are discussed in terms of the intracellular localization of a possible submembranous contractile system involved in motile activities such as cell locomotion.     

Isolation and characterization of tropomyosin-containing microfilaments from cultured cells

We have developed a new method for the rapid isolation of tropomyosin-containing microfilaments from cultured cells using anti-tropomyosin monoclonal antibodies. Anti-tropomyosin monoclonal antibodies induce the bundle formation of microfilaments, which can be easily collected by low speed centrifugation. Electron microscopic studies of the isolated microfilaments show periodic localization of tropomyosin along the microfilaments of nonmuscle cells with a 33-34 nm repeat. Furthermore, the isolated microfilaments have the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin to almost the same extent as skeletal muscle F-actin (filamentous actin). This microfilament isolation method is applicable to a variety of cell types, including REF-52 cells (an established rat embryo line), L6 myoblasts, 3T3 fibroblasts, Chinese hamster ovary cells, baby hamster kidney (BHK-21) cells, mouse neuroblastoma cells, gerbil fibroma cells, and chicken embryo fibroblasts. Sodium dodecyl sulfate-polyacrylamide gel analysis shows that, in addition to actin, microfilaments isolated from REF-52 cells contain five species of tropomyosin with apparent Mr = 40,000, 36,500, 35,000, 32,400, and 32,000, alpha-actinin, and as yet unknown proteins with apparent Mr = 83,000 and 37,000. The molar ratio of total tropomyosin (dimer) to actin in the isolated microfilaments is 1:8. The patterns of these multiple forms of tropomyosin were found to change when REF-52 cells were transformed with SV40 or adenovirus type 5.     

Cytochalasin B-induced redistribution of cytokeratin filaments in PtK1 cells

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.     

Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.     

Improved negative staining of microfilament arrangements in detergent-extracted Physarum amoeboflagellates

A motile, lamellipodium-like structure, the ridge, forms as amoeboflagellate cells of Physarum polycephalum release from a substratum and begin swimming in fluid. Actin microfilaments form a distinct laminar core within the ridge; they are seen as a sparse, disordered meshwork in cytoskeletons prepared by conventional methods using uranyl acetate negative staining [10]. Preservation and visualization of these filaments and their arrangements improved considerably when cytoskeletons were imaged with phosphotungstic acid buffered with ammonium hydroxide (PTA(NH4]. Microfilaments within ridge cytoskeletons were found to form loose bundles and criss-crossing, 'meshwork' arrays several layers deep. Differences could be detected in morphology and detailed arrangement of microfilaments within cytoskeletons prepared in the presence of phalloidin. PTA(NH4) may be useful for studies of cytoskeletal elements and their rearrangements in dynamic, motile regions of cells.     

The role of microfilaments in the spreading process of JTC-12 cells

In 5 μg/ml cytochalasin B (CB), spreading of JTC-12 cells over the substratum occurred to some extent, but an almost complete inhibition was seen in 10 μg/ml CB, except for extrusion of thin processes. Formation of microfilament bundles beneath the adhesive surface was correlated with the grade of spreading. Treatment of spreading cells with 10 μg/ml CB caused a retraction of the peripheral cytoplasm or inhibited further spreading and concurrently disintegrated the microfilament bundles. Thus, the circular bundles of the microfilaments inside the cell outline probably enable the concentrical spreading of JTC-12 cells by advancing and consolidating the peripheral 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.     

Glycolipid Content of Vesicular Stomatitis Virus Grown in Baby Hamster Kidney Cells

Vesicular stomatitis virions grown in baby hamster kidney (BHK-21-F) cells were found to contain hematoside (neuraminosyl-galactosyl-glucosyl-ceramide). This ganglioside, which was the only detectable glycolipid in the virion, is also the only glycolipid found in significant amount in BHK-21-F cells. Approximately 87% of the total neuraminic acid in the virion was found to be linked to protein and 13% to lipid.     

THE DISTRIBUTION, ULTRASTRUCTURE, AND CHEMISTRY OF MICROFILAMENTS IN CULTURED CHICK EMBRYO FIBROBLASTS

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.     

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.     

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

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

Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.

Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.     

FIBRILLAR DIFFERENTIATION IN A MICROPLASMODIUM OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

The motility of Physarum polycephalum microplasmodia depends upon the conditions under which they are cultured. To investigate the relation between protoplasmic streaming and filamentous structures observed in the cytoplasm, microplasmodia were collected from shaken cultures, agar plates and shaken cultures of the organism which had previously been plate-cultured.
No sign of streaming could be found in materials in shaken culture, even in those which were shaken after they had once been motile on an agar plate. The immotile microplasmodia in both cases failed to contain any filamentous structures.
Microplasmodia on agar plates were motile, showing vigorous peripheral movements (projection of pseudopods) and inner protoplasmic streaming. In the motile organisms two types of filamentous structures were observed: loose networks just inside the plasma membrane of rounded pseudopods with smooth surfaces; and compact, straight bundles beneath the pseudopods or in much deeper locations.     

FINE STRUCTURE OF CALONEIS AMPHISBAENA (BACILLARIOPHYCEAE)1

The structure of the motile pennate diatom Caloneis amphisbaena Cleve is described, with emphasis on the lateral, lobed pyrenoid with neither a limiting membrane nor penetration by thylakoids, an interphase nucleus with centers of condensed chromatin, paired dictyosomes, and mitochondria cradled within the chambers of the valve. Microfilaments forming two bundles which lie beneath each raphe slit are of the same size and appearance as actin microfilaments associated with other motile systems.     

An intermediate filament-associated protein, p50, recognized by monoclonal antibodies

Monoclonal antibodies (mAB) were raised to be used as probes to identify cytoplasmic components associated with intermediate filaments (IF). Four hybridomas (B27, B76, B78, and B100) secreting mAB were generated by fusing mouse myeloma cells with the spleen cells of mice immunized intraperitoneally with Triton-high salt insoluble materials from BHK-21 cells. This insoluble material consists mostly of IF, a small number of microfilaments, and some polyribosomes. Biochemical studies show that the Triton-insoluble materials contain many proteins, including vimentin (decamin) and desmin. Immunofluorescence microscopy of BHK-21 cells stained with the four mAB showed that these mAB decorate the IF in a dotted pattern. Double staining with polyclonal antibody to vimentin confirmed the reactivity of the mAB with the IF. These mAB also stained the vimentin-containing filament system in a variety of other cells including epithelial cells (PTK1 and HeLa) and cells of astroglial origin. Histological studies showed that mAB-B100 stained many types of tissue including epidermis, smooth muscle, and subdermis pericytes, but not the white matter nor the gray matter of the cerebellum and spinal cord. Immunoelectron microscopy with colloidal gold has shown that the mAB-B100 decorated the IF in clusters or aggregates around proteinaceous materials associated with the filaments. Results of immunoprecipitation indicate that mAB-B100 reacted with a protein of 50,000 daltons. These findings suggest that the mAB-B100 we have developed recognizes one of the many components of what appears to be an integrated cytoskeletal structure connected with intermediate filaments.     

紫萼花粉粒和花粉管中微丝的超微结构

用常规化学固定和化学固定前用鬼笔环肽处理两种电镜样品制作技术,分别研究了紫萼[Hosta venteicosa (=H.coerulea]成熟花粉粒和幼花粉管中的微丝的超微结构。结果表明,在常规电镜固定中花粉粒中的微丝能保存,但在花粉管中的则遭受破坏。用鬼笔环肽处理后化学固定的方法,微丝在花粉管中能良好地保存。在花粉粒中平行的微丝形成束,表现为具分布的特点,即限于分布在它们功能的区域,并且微丝束经常紧密地与营养核贴近。在幼花粉管中微丝束表现为在线粒体、质体、内质网、小泡和小液泡的表面通过,并常常与脂体紧密联结。这些现象表明在花粉萌发和花粉管生长时,微丝与营养核及与其它细胞器的运动之间存在某些联系的迹象。     

Role of Interferon in the Propagation of MM Virus in L Cells

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.     

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.