Transfections of primary muscle cell cultures with plasmids coding for GFP linked to full-length and truncated muscle proteins.
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Animals↗
Biomedical subjects
J W Sanger
Publications and source records attributed to J W Sanger.
Unusual cleavage furrows in vertebrate tissue culture cells: insights into the mechanisms of cytokinesis.
In cultures of the epithelial cell lines, PtK2 and LLC-PK, some cells assume unusually large flattened morphologies and, during cell division, produce unusual cleavage furrows. We have microinjected some of these large cells with fluorescent actin and myosin probes to determine how the cell's shape and the position of its mitotic spindle affect the deposition of actin and myosin in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent nondaughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured epithelial cells conformed to the furrows seen when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another [Rappaport, 1961]. The recruitment of contractile proteins and the formation of furrows between adjacent centrosomes was a function of the distances between them. When adjacent centrosomes were positioned too close together neither contractile protein recruitment nor furrow formation occurred. If a normal-sized spindle was present in a very large cell, fibers of contractile protein assembled in the cortex above the former metaphase plate but they did not extend to the cell periphery, resulting in an inhibition of cytokinesis. In all injected cells, the recruitment of actin and myosin to the cell surfaces could first be detected at mid-anaphase before there was any visible sign of furrowing. Our results suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells. The results are consistent with a model in which (1) the positions of the centrosomes and their linearly connected microtubules determine the position for the assembly of the cleavage furrow, and (2) the signal arrives at the surface within a few minutes after the initiation of anaphase. We speculate that an interaction of the ends of microtubules from adjacent centrosomes with the cell surface promotes a clustering of integral membrane protein(s) that interact with and target contractile proteins to a position midway between centrosomes where furrowing occurs.
Animals↗
Sites of monomeric actin incorporation in living PtK2 and REF-52 cells.
The purpose of this study was to analyze where monomeric actin first becomes incorporated into the sarcomeric units of the stress fibers. We microinjected fluorescently labeled actin monomers into two cell lines that differ in the sarcomeric spacings of alpha-actinin and nonmuscle myosin II along their stress fibers: REF-52, a fibroblast cell line, and PtK2, an epithelial cell line. The cells were fixed at selected times after microinjection (30 s and longer) and then stained with an alpha-actinin antibody. Localization of the labeled actin and alpha-actinin antibody were recorded with low level light cameras. In both cell types, the initial sites of incorporation were in focal contacts, lamellipodia and in punctate regions of the stress fibers that corresponded to the alpha-actinin rich dense bodies. The adherent junctions between the epithelial PtK2 cells were also initial sites of incorporation. At longer times of incorporation, the actin fluorescence extended along the stress fibers and became almost uniform. We saw no difference in the pattern of incorporation in peripheral and perinuclear regions of the stress fibers. We propose that rapid incorporation of monomeric actin occurs at the cellular sites where the barbed ends of actin filaments are concentrated: at the edges of lamellipodia, the adherens junctions, the attachment plaques and in the dense bodies that mark out the sarcomeric subunits of the stress fibers.
Actins↗
Occurrence of fibers and their association with talin in the cleavage furrow of PtK2 cells. Video supplement.
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Actin Cytoskeleton↗
Interactions of Listeria monocytogenes with infected host cells. Video supplement.
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Actinin↗
Myofibrillogenesis visualized in living embryonic cardiomyocytes.
Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, alpha-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing alpha-actinin. The GFP-alpha-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of alpha-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils-i.e., the precursors of mature myofibrils.
Actinin↗
Premyofibrils in spreading adult cardiomyocytes in tissue culture: evidence for reexpression of the embryonic program for myofibrillogenesis in adult cells.
Do adult cardiomyocytes use the same pathways hypothesized for the formation of myofibrils in embryonic cardiomyocytes in tissue culture. [Rhee, et al., Cell Motil. Cytoskeleton 28:1-24, 1994]? Premyofibrils in embryonic cardiomyocytes are composed of short sarcomeric units of alpha-actinin (Z-bodies) and actin filaments held together by short nonmuscle myosin IIB filaments. Premyofibrils are believed to be transformed into nascent myofibrils by their capture of muscle-specific myosin II filaments aligned in aperiodic arrays. Nascent myofibrils are thought to transform into mature myofibrils by the loss of nonmuscle myosin IIB, the fusion of the Z-bodies into Z-bands, and the periodic alignment of muscle myosin II filaments into A-bands. Freshly isolated cat and rat adult cardiomyocytes placed in tissue culture lack premyofibrils and nascent myofibrils. Adult cardiomyocytes spreading in culture reinitiate the synthesis of nonmuscle myosin IIB. Moreover, patterns similar to the proposed embryonic myofibrillar program first detected in spreading chick embryonic hearts were also detected in these spreading adult mammalian cardiomyocytes. The isolated adult cardiomyocytes begin to spread after 1 day in culture by sending out lamellipodia. When these cells are injected with fluorescently labeled alpha-actinin, linear arrays of short spacings of beaded alpha-actinin bodies are detected in the spreading edges of the adult cardiomyocytes. These dense bodies (Z-bodies) stain positively for the same sarcomeric-specific isoform of alpha-actinin that is in the Z-bands of mature sarcomeres. These linear arrays of alpha-actinin-containing Z-bodies have other characteristics of premyofibrils and are detected only in the spreading regions of the cells. Thus, these premyofibrils at the edges of the spreading adult cardiomyocytes stain positively for nonmuscle myosin IIB but negatively for muscle-specific myosin II. Initially, no vinculin is associated with any parts of the premyofibrils in the spreading regions of the early spreading cardiomyocytes. However, later, vinculin is found to be associated with the ends of the premyofibrils. Fibers that stain solidly for muscle-specific myosin II (i.e., nascent myofibrils) are localized between the peripheral premyofibrils and the centrally positioned, mature myofibrils. It is suggested that the puzzling ability of cardiomyocytes in hypertrophic hearts to reinitiate the synthesis of fetal sarcomeric proteins may be related to the reinitiation of the embryonic premyofibril program for myofibrillogenesis.
Animals↗
Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils.
Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.
Actin Cytoskeleton↗
An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.
Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.
Amino Acid Sequence↗
Zeugmatin is part of the Z-band targeting region of titin.
Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.
Animals↗
Living adult rat cardiomyocytes in culture: evidence for dissociation of costameric distribution of vinculin from costameric distributions of attachments.
Adult rat cardiomyocytes were placed in tissue culture to determine the relationships of their vinculin positive costameres, their attachments associated with the costameres, the fate of their myofibrils. The costameric structures were detected using interference contrast microscopy and the visualization of the fluorescently labeled vinculin and alpha-actinin molecules. The cardiomyocytes isolated from the heart retained their myofibrils upon attachment to the cell surfaces. One group of cells then rounded up, only to respread after 6 days in culture. These cells initially demonstrated costameric distributions of attachments and vinculin. These relationships were lost during the rounding-up process only to be regained as the cells respread. The second group of freshly isolated cardiomyocytes did not round up but began to spread on the substratum by sending out lamellipodia from their rectangularly shaped body. These newly cultured cardiomyocytes initially exhibited costameric distributions of close attachments detected by interference microscopy. Over the next 3 days although the cells remain attached to the substratum, the costameric attachments were gradually lost. Nevertheless, when similar cells were injected with fluorescently labeled vinculin, costameric distributions of vinculin could be detected in the absence of costameric attachments. Cardiomyocytes, injected with fluorescent alpha-actinin, revealed that during the first few days in culture the existing myofibrils disassembled from the edges of the cell towards the middle. The center group of myofibrils was retained as the cells began to spread. Our observations of living cells support the hypothesis that proteins in addition to vinculin are needed for cardiomyocytes to generate costameric attachments to the cell surfaces. We speculate that the ability of the vinculin-attached Z-lines of adult cardiomyocytes to dissociate from the extracellular matrix may aid in the remodeling of the adult heart in the repair process after myocardial infarction and also in stress induced hypertrophic growth.
Actins↗
Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin.
Zeugmatin is a muscle specific protein discovered by Maher et al. [1985: J. Cell Biol. 101:1871-1883] to be in Z-Bands of muscle and in the dense bodies of smooth muscle. Maher et al. [1985] generated a zeugmatin specific monoclonal antibody, McAb20, and then used immunoaffinity chromatography to isolate a 600-800 kD protein. During myofibrillogenesis of embryonic cardiac muscle, zeugmatin is detected in fully formed Z-bands in the mature myofibrils but not in the Z-bodies of premyofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24]. Rhee et al. [1994] have postulated that zeugmatin may be responsible for the fusion of the alpha-actinin containing Z-bodies to form the solid Z-Bands of the mature myofibrils. The current studies were undertaken to characterize the properties of zeugmatin. The McAb20 was used to probe a chicken heart lamba gt11 expression library, and three unique positive clones of 1.1, 1.4, and 1.7 kB were isolated. These were inserted into pcDNA3, sequenced, and assembled into a 1.8 kB ORF. A 60% identity with N-terminal region of the human cardiac titin sequence was revealed at the amino acid level. This region of the 1.8 kB zeugmatin sequence is located entirely in the Z-band region of the human cardiac titin molecule. The 1.1 kB clone of zeugmatin was subcloned into pTrcHisC and expressed in bacteria. Bacterial lysates were prepared and run over nickel columns to isolate a 46 kD fusion protein. This fusion protein formed a complex with purified alpha-actinin that could be immunoprecipitated with the zeugmatin specific antibody, McAb 20. The 1.1 kB sequence was transfected into non-muscle cell lines, PtK2 and REF. Twenty-four hours after transfection, the 46 kD zeugmatin peptide, not present in control non-muscle cells, was localized in focal adhesions and in a punctate pattern along the stress fibers. Double immunofluorescence staining revealed that zeugmatin colocalized with the alpha-actinin in the dense bodies and focal contacts of the stress fibers. At longer time points, as the transfected cells accumulated more truncated zeugmatin molecules, the cells lost adhesion plaques and stress fibers, and became detached from the substratum. Our results indicate the zeugmatin is part of the titin molecule that is located within the Z-band and that this section of the titin molecule anchors the actin crosslinking alpha-actinin molecules.
Actinin↗
Novel form of actin-based motility transports bacteria on the surfaces of infected cells.
Enteropathogenic Escherichia coli (EPEC) attach to cells (attachment) lining the intestine and induce a decrease in the number of the cells' microvilli (effacement). This attachment and effacement is followed by diarrhea, which may be explained, at least in part, to the loss of microvilli and the decreased ability of the infected cells to absorb fluids. EPEC also attach to the surfaces of a number of cultured cells including CaCo-2, LLC-PK, and PtK2 cells. The extracellular, attached EPEC induce filaments of actin to form in the cytoplasm just underneath the EPEC surface attachment sites. Beneath some of the attached EPEC, the actin filaments become organized into membrane encased columns that extend up to 6 micrometers above the cell surface creating "pedestals" on which the EPEC rest. The raised pedestals can be readily observed in stereo pairs taken using the Intermediate Voltage Electron Microscope. The concentration of non-muscle isoforms of myosin II and tropomyosin near the base of the pedestals suggests a similarity of these structures to brush border microvilli. Video microscopy indicates that these EPEC pedestals can bend and undulate, alternately growing longer and shorter while remaining tethered in place on the cell surface. Some of the attached EPEC also translocate along the cell surface, reaching speeds up to 0.07 micrometers/sec. Both types of movement are inhibited by cytochalasin D, indicating that actin polymerization in the pedestals is required for the motility of EPEC on the host cell surface. In this respect, EPEC motility on host cells resembles the intracellular motility of Listeria, but there are differences in the actin filament bundles induced by the two different bacteria. The most obvious one is the interposition of the cell membrane between EPEC and the actin filaments in the pedestal in contrast to the close apposition of actin filaments to Listeria. The intensity of fluorescence of rhodamine phalloidin is nearly uniform along most of the length of the pedestals indicating a constant number of actin filaments, whereas the fluorescence intensity decreases along the length of Listeria tails reflecting the disassembly that occurs all along the tails. Epec's movements may be a hybrid of Listeria filopodia and Aplysia inductopodia movements. This paper is the first report of a microbe attached to the extracellular surface of an infected cell propelled by an intracellular actin polymerization-dependent mechanism.
Actins↗
Listeria monocytogenes intracellular migration: inhibition by profilin, vitamin D-binding protein and DNase I.
Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail-like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthens, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell-to-cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial-actin tail interface. We have studied the consequences of microinjecting three different actin monomer-binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11-22 microM) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer-sequestering proteins, Vitamin D-binding protein (DBP; 1-2 microM) and DNase I (6-7 microM) completely block bacterial-induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10-20 min) indicating that the mechanisms by which these proteins interfere with Listeria-induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer-binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 microM). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria-induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer-binding proteins.
Actinin↗
Organization and structure of actin filament bundles in Listeria-infected cells.
During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria-column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria-associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: 1) the surface of the bacteria, 2) the cytoplasm next to the bacteria, 3) the outer shell of the actin column, and 4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells.
Actins↗
Increasing intracellular concentrations of thymosin beta 4 in PtK2 cells: effects on stress fibers, cytokinesis, and cell spreading.
Thymosin beta 4 (T beta 4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of increasing T beta 4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of T beta 4 at a concentration (approximately 28 microM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular T beta 4 concentrations were increased by three different methods: 1) microinjection of an RSV vector containing a cDNA for T beta 4; 2) transfection with the same vector; and 3) microinjection of purified T beta 4 protein. The plasmid coding for T beta 4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure T beta 4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, similar microinjection of pure thymosin beta 4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow regressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these T beta 4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process.
Actinin↗
Occurrence of fibers and their association with talin in the cleavage furrows of PtK2 cells.
PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows.
Actin Cytoskeleton↗
The premyofibril: evidence for its role in myofibrillogenesis.
When cardiac muscle cells are isolated from embryonic chicks and grown in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of alpha-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded alpha-actinin staining range from 0.3 to 1.4 microns in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 microns) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils.
Actinin↗