Intermediate filaments in skeletal and cardiac muscle tissue in embryonic and adult chicken.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S J Singer
Publications and source records attributed to S J Singer.
Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues.
A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.
Animals↗
The reorientation of the Golgi apparatus and the microtubule-organizing center in the cytotoxic effector cell is a prerequisite in the lysis of bound target cells.
This investigation is concerned with the detailed mechanisms of cytotoxicity, and in particular, with early cell surface and intracellular events that occur upon the binding of a cytotoxic effector cell to its susceptible target. In earlier studies, we have shown by immunofluorescence microscopy that when cloned natural killer (NK) and cytolytic T lymphocyte (CTL) cells bind to susceptible target cells, a rapid and coordinate reorientation of the perinuclear Golgi apparatus and the microtubule-organizing center (MTOC) occurs inside the effector cell so that the two organelles face the bound target. It was proposed that the purpose served by this reorientation is to direct Golgi-derived secretory vesicles, containing one or more cytotoxic components, to the area of target cell binding. In order to establish more firmly that this reorientation of the two organelles is an essential early event in cytotoxicity, we have performed three different types of experiments. 1) Upon binding cloned effector cells to susceptible targets in the presence of Mg+2 but absence of Ca+2, conditions in which no killing occurs, we found that no reorientation of the MTOC in the effector cell is induced; however, the addition of CA+2 to these cell couples results in a rapid MTOC reorientation. 2) With a lysis-defective subclone derived from a cytolytic NK clone, binding to target cells did not induce an MTOC reorientation in the defective killer cell. 3) In multitarget conjugates formed with single CTL, the MTOC in the CTL was oriented to face that one target cell which was in the process of lysis at the time. These results, together with our earlier findings, strongly indicate that a Golgi/MTOC reorientation inside the target-bound effector cell is a pre-requisite for the effective lysis of the target. They also reveal the existence, previously unrecognized, of a specific signaling mechanism from the viable target cell to the effector cell, which must be involved in the triggering of this Golgi/MTOC reorientation. These conclusions are consistent with the proposal that a polar cytotoxic secretory mechanism is responsible for target cell lysis.
Animals↗
Structural changes in membranes produced by the binding of small amphipathic molecules.
In their interactions with membranes, amphipathic small molecules exhibit detergent-like properties. At sufficiently high concentrations (above their critical micelle concentrations, if they form micelles), they substantially dissolve membranes. At lower concentrations, between maximally antihemolytic and lytic, we show here that the amphipaths significantly perturb membrane structure. Each of six small-molecule amphipaths was shown by hygroscopic desorption filtration to induce the extraction of small but significant amounts of membrane components, partly in the form of vesicular fragments, from red blood cell membranes. These extracts were enriched in the lipid to protein ratio as compared to the intact membrane, and the protein composition was highly unrepresentative. A similar set of extractions from sarcoplasmic reticulum membranes was induced by the six amphipaths. We conclude that small-molecule amphipaths, at concentrations lower than lytic, promote gross redistributions of components in the plane of a membrane that result in the observed extractions.
Animals↗
An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections.
Ultrathin frozen sections are ideal substrates with which to carry out immunolabeling experiments in electron microscopy. However, the ultrastructural delineation in positively stained frozen sections has not been as detailed as in conventionally osmium-stained and plastic-embedded sections. We now describe a simple technique in which immunolabeled ultrathin frozen sections are subsequently treated with osmium tetroxide, dehydrated, and then embedded in plastic by impregnation with a monomer to the thickness of the section, followed by polymerization of the monomer. By this technique ultrastructural definition as good as that of conventional plastic sections is achieved, while the high density and specificity of immunolabeling characteristic of ultrathin frozen sections are retained.
Animals↗
Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.
Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubules reflects some type(s) of linkage(s) between them and that such linkages may play an important role in the location and intracellular transport of lysosomes inside cells.
Animals↗
Distributions of vimentin and desmin in developing chick myotubes in vivo. I. Immunofluorescence study.
Antibodies against chicken erythrocyte vimentin and gizzard desmin were affinity purified and then cross-absorbed with the heterologous antigen. They were used to study the in vivo distributions of these proteins in developing and mature myotubes by immunofluorescence microscopy of 0.5-2-micron frozen sections of iliotibialis muscle in 7-21-day chick embryos, neonatal and 1-d postnatal chicks, and adult chickens. The distributions of vimentin and desmin were coincidental throughout the development of myotubes, but the concentration of vimentin was gradually reduced as the myotubes matured and became largely undetectable at the time of hatching. The process of confining these proteins to the level of Z line from the initial uniform distribution occurred subsequent to the process of bringing myofibrils into lateral registry: in-register lateral association of several myofibrils was occasionally seen as early as in 7-11-d embryos, whereas the cross-striated immunofluorescence pattern of desmin and vimentin was only vaguely discerned in myotubes of 17-d embryos, just 4 d before hatching. In some myotubes of 21-d embryos, myofibrils were in lateral registry as precisely as in adult myofibers but desmin was still widely distributed around Z line in an irregular manner. Nevertheless, in many other myotubes of prenatal or neonatal chicks, desmin became confined to the level of Z line in a manner similar to that seen in adult myofibers, thus essentially completing its redistribution to the confined state of adult myofibers in coincidence with the time of hatching. In extracts from iliotibialis and posterior latissimus dorsi muscles of adult chickens, we detected a hitherto unidentified protein that was very similar to vimentin in molecular weight but did not react with our antivimentin antibody. We discuss the possibility that this protein was confused with vimentin in the past.
Aging↗
Associations of elements of the Golgi apparatus with microtubules.
The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.
Animals↗
Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.
We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.
Animals↗
A 200-kd protein isolated from the fascia adherens membrane domains of chicken cardiac muscle cells is detected immunologically in fibroblast focal adhesions.
On the premise that the fascia adherens of cardiac muscle cell intercalated disk membranes is a structure that is closely homologous to the focal adhesions formed by fibroblasts, a fascia adherens preparation was isolated from chicken cardiac muscle, and was analyzed for its protein composition. A prominent 200-kilodalton (kd) protein was purified from the fascia preparation and shown to be antigenically unrelated to several previously characterized cytoskeletal proteins, including cardiac myosin and vinculin. With monospecific antibodies to the 200-kd protein, an identical or closely similar intracellular protein was shown to be associated with the focal adhesion plaques of fibroblasts.
Animals↗
Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets.
In cell couples formed between a cloned murine natural killer (NK) cell and either of two target cells, we have obtained evidence by immunofluorescence observations for a rapid coordinate repositioning of two organelles, the microtubule organizing center and the perinuclear Golgi apparatus, inside the NK cell to face the region of contact with the target cell. With microtubule-disrupting drugs, which also cause a dispersion of the Golgi apparatus throughout the periphery of the NK cell, it was found that target cell lysis is reversibly inhibited. It is proposed that the coordinate repositioning of the two organelles serves the function of directing secretory vesicles derived from the Golgi apparatus to the bound target cells, the secretory vesicles containing components that induce target cell lysis.
Animals↗
Membrane insertion at the leading edge of motile fibroblasts.
We are concerned with the mechanisms involved in the directed migration of eukaryotic cells. Previously we found that, inside cells at the edge of an experimental wound, the Golgi apparatus and the microtubule-organizing center were rapidly repositioned forward of the nucleus in the direction of subsequent cell migration into the wound. This repositioning was proposed to serve the purpose of introducing new membrane mass at the leading edge of the cell, by directing Golgi apparatus-derived vesicles bound for the plasma membrane to that edge. We now provide evidence to support this proposal. Cultured fibroblastic cells at the edge of a wound were infected with a temperature-sensitive mutant (0-45) of vesicular stomatitis virus. It is known that the G-protein, an integral membrane protein of the virus, is synthesized and remains in the rough endoplasmic reticulum at the nonpermissive temperature, but when the infected cells are shifted to the permissive temperature, the G-protein moves through the Golgi apparatus to the plasma membrane. By immunofluorescence microscopy, we here show that the first appearance of the G-protein at the cell surface corresponds to the leading edge of the motile cell. These observations are incorporated into a coherent scheme for the mechanisms involved in cell migration.
Animals↗
Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle.
We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the T-tubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system.
Animals↗
Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle.
We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.
Actinin↗
Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells.
An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.
Animals↗
Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts.
Triple-immunofluorescence experiments with antibodies to cytochrome c oxidase, tubulin, and vimentin have been used to immunolabel the mitochondria, microtubules, and intermediate filaments inside the same cultured fibroblasts. In particular, fibroblasts were immunolabeled after they had either been transformed by infection with Rous sarcoma virus or given long-term treatment with cycloheximide. These treatments induced redistribution of the intermediate filaments into a perinuclear arrangement, segregated away from the microtubules, which remained extended to the cell periphery. In such cells, many labeled mitochondria were observed to be codistributed with the peripherally located microtubules. From these results, we infer that an association, probably involving some type of chemical linkage(s), between mitochondria and microtubules exists in these cells that is independent of the intermediate filaments.
Animals↗
Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide.
Antisera were raised against a synthetic peptide (src-c) containing the six COOH-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus (RSV). Antibodies specific for the src-c peptide were purified by affinity chromatography and then used to study the location of p60src in transformed cells. The distribution of p60src was compared to that of vinculin, a candidate cytoskeletal substrate of p60src, by indirect double immunofluorescence microscopy. In RSV-transformed rat, mouse, and chicken cells, an extensive codistribution of p60src with vinculin was observed. Both proteins were concentrated in the few remaining focal adhesion plaques, in transformation-induced rosette clusters at the ventral cell surface, and in cell-cell contact areas. In addition, antibodies to both proteins stained the cytoplasm diffusely. In all cells examined, the immunofluorescent staining patterns produced by antibodies to the src-c peptide were indistinguishable from those obtained by immunolabeling of p60src with sera from RSV-infected tumor-bearing rabbits. The excellent agreement of the results obtained with two completely independent antibody preparations indicates strongly that the observed immunolabeling patterns correctly define the intracellular distribution of p60src. The significance of the intracellular location of p60src to the transforming activities of the protein is discussed.
Animals↗
On the nature of crossreactions observed with antibodies directed to defined epitopes.
Antibodies directed against a synthetic peptide (src-c) containing the six carboxyl-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus, recognize p60src. However, when used at sufficiently high concentrations they also react with a number of constituents of untransformed cells. These reactions can be completely inhibited by src-c peptide. Crossreactivities are to different components in cells from different species and cannot be attributed to p60c-src, the ubiquitous cellular homologue of p60src. By indirect immunofluorescence microscopy and immunochemical techniques we have identified three cytoskeletal proteins, myosin, tubulin, and vimentin, as well as an unknown intranuclear antigen, as major targets of anti-src-c antibodies in different untransformed cells. These crossreactivities probably reflect identities or similarities in the amino acid sequence of the immunogenic peptide and segments of the otherwise unrelated crossreactive proteins. These findings are discussed with respect to the interpretation of crossreactivities that are occasionally observed with anti-peptide sera and with monoclonal antibodies.
Amino Acid Sequence↗