Selective binding of antibody against gizzard 10-nm filaments to different cell types in myogenic cultures.

Antibody against the intermediate-sized filaments from gizzard smooth muscle was used to determine the presence or absence of reacting 10-nm filaments in different cell types. The antibody against gizzard 10-nm filaments reacted with filaments in cultured smooth muscle cells, skeletal myotubes and postmitotic skeletal myoblasts. It did not bind to the 10-nm filaments present in replicating presumptive myoblasts and fibroblasts, or the 10-nm filaments in spinal ganglion cells.     

Epstein-Barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs.

Peripheral blood lymphocytes from normal individuals and patients with autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis were infected with Epstein-Barr virus, and the culture supernatants were tested for autoantibodies that reacted with normal tissues. Between 58 and 86% of Epstein-Barr virus-transformed cultures produced immunoglobulin M antibodies, and between 9 and 24% of the transformed cultures produced immunoglobulin G antibodies that reacted with normal tissues. Ten Epstein-Barr virus-transformed clones secreting human immunoglobulin M monoclonal autoantibodies were isolated. Four of these monoclonal autoantibodies were studied in depth and found to react with antigens in multiple organs, including thyroid, pancreas, stomach, smooth muscle, and nerves. It is concluded that Epstein-Barr virus can trigger the production of autoantibodies without infecting the target cells to which the autoantibodies are directed.     

Tumour-specific Antibodies in Human Malignant Melanoma and their Relationship to the Extent of the Disease

Biopsy specimens and sera were obtained from 103 melanoma patients. Autoantibodies were demonstrated by (1) complement-dependent cytotoxicity of autologous melanoma cells in short-term culture; (2) complement-dependent inhibition of ribonucleic acid synthesis; (3) immunofluorescent staining of the cytoplasm of killed melanoma cells and of the surface membrane of viable melanoma cells. Over one-third of the sera studied had antibodies to autologous melanoma cells. Although for technical reasons all three tests could not be performed with the cells from every melanoma, whenever multiple testing was possible there was complete concordance. The autoantibodies were virtually confined to patients in whom the disease was not widely disseminated, and over 80% of such patients had positive sera. In a limited number of patients who have been followed autoantibodies disappeared as the disease progressed to become widely disseminated. Two patients with generalized disease developed autoantibodies following inoculation by their own irradiated tumour cells.Two types of autoantibodies were recognized: one, active against antigen(s) in the cell surface membrane, was specific for each tumour—that is, only the autologous serum reacted—and was concerned in the cytotoxic activity; the other reacted with cytoplasmic antigens which appeared to be present in most or all melanoma cells.     

Dense bodies and actin polarity in vertebrate smooth muscle

The arrangement of cytoplasmic dense bodies in vertebrate smooth muscle and their relationship to the thin filaments was studied in cells from rabbit vas deferens and portal vein which were made hyperpermeable (skinned) with saponin and incubated with myosin subfragment 1 (S-1). The dense bodies were obliquely oriented, elongated structures sometimes appearing as chains up to 1.5 microns in length; they were often continuous across the cell for 200 to 300 nm and were interconnected by an oblique network of 10-nm filaments. The arrowheads, formed by S-1 decoration of actins, which inserted into both the sides and ends of dense bodies, always pointed away from the dense body, similar to the polarity of the thin filaments at the Z- bands of skeletal muscle. These results show that the cytoplasmic dense bodies function as anchoring sites for the thin filaments and indicate that the thin filaments, thick filaments, and dense bodies constitute a contractile unit.     

Myosin and actin containing cells in the human postnatal thymus. Ultrastructural and immunohistochemical findings in normal thymus and in myasthenia gravis.

Samples of normal human thymus of different ages (4-63 years old) were studied by immunofluorescence microscopy (using antibodies to smooth muscle myosin, to actin from the chicken gizzard, and antibodies to myosin from human striated muscle) as well as by routine electron microscopy. Thymus tissue from myasthenia gravis patients was also investigated for comparative reasons. Epithelial cells reacted with anti-smooth, but not with anti-striated muscle myosin, whereas myoid cells reacted with antibodies to striated, but not to smooth muscle myosin. Both epithelial and myoid cells displayed a strong immunoreactivity with antiactin. Corresponding to this immunoreactivity, both cell types contained bundles of thin, actin-like filaments. Myoid cells occurred in the rounded and elongated variety, and they were a normal constituent of all thymuses investigated in this study. Ultrastructurally, this non-innervated, striated muscle-like cell type possessed bundles of thin and thick filaments as well as Z lines in a rather disorganized arrangement, resembling striated muscle after denervation or various other pathologic conditions. There were no overt differences in the number and structure of myoid cells between healthy and myasthenic patients.     

Development and differentiation of the rat epididymis. I: ultrastructural aspects of the peritubular zone

We investigated the ultrastructural aspects of the peritubular cells of epididymis and their development from birth to adult age. At birth the peritubular zone consisted of polygonal cells which did not differ from other interstitial cells. Cytoplasmic filaments were visible in the cells of the inner layer at day 6. From day 22 the peritubular cells reached the adult aspect. The peritubular cells in the rat epididymis had aspects similar to those of peritubular smooth muscle cells of rat testis, with a more precocious appearance of cytoplasmic filaments. This finding concurs with the observed precocious contractility of epididymis.     

Biochemical and immunological heterogeneity of 100 A filament subunits from different chick cell types.

The 100 A filament subunit proteins of chick fibroblasts and gizzard smooth muscle were compared. These proteins are major cellular components in these cell types, constituting up to 98% of the cell's total protein. Co-electrophoresis of cytoskeletal fractions of fibroblasts and smooth muscle revealed that the subunit proteins differed in their molecular weights: 58,000 daltons in fibroblasts and 55,000 daltons in smooth muscle. Cytoskeletal fractions from other cell types were also examined: chondroblasts contained the 58,000 dalton subunit, and cytoskeletons of skeletal muscle and cardiac muscle contained both 55,000 and 58,000 dalton proteins. Chick skin and rat kangaroo Pt K2 cells had more complex subunit patterns which resemble prekeratin. The peptide patterns resulting from proteolytic digestion of the 58,000 dalton protein of fibroblasts, the 55,000 dalton proteins of smooth muscle and PT K2 cells, and chick brain tubulin differed from one another. Two-dimensional electrophoresis of reconstituted gizzard smooth muscle 100 A filaments showed the 55,000 dalton subunit to be composed of two major components, differing in their isoelectric points. Antibodies prepared against electrophoretically purified 55,000 dalton subunit protein reacted in immunodiffusion against the original smooth muscle antigen and cytoskeletal fractions from skeletal and cardiac muscle, but not from fibroblasts, brain, liver, or skin cells. A specific antigenic determinant common to subunit proteins in smooth, skeletal, and cardiac muscle, is therefore indicated. A previously described antibody against fibroblast subunit protein reacted weakly against smooth muscle filament protein in immunodiffusion revealing the presence of a common antigenic determinant between the two subunit proteins. These data demonstrate striking antigenic and primary structural differences in 100 A filament subunits from even such closely related cell types as fibroblasts on the one hand and muscle cells on the other.     

The distribution of desmin (100 A) filaments in primary cultures of embryonic chick cardiac cells.

Using antibodies to desmin, the major component of the 100Å-filaments from smooth muscle cells, we studied by indirect immunofluorescence the distribution of this protein in primary cultures of embryonic chick cardiac cells. We show that desmin is a component of cytoplasmic filamentous structures which comprise a network distinct from actin filament bundles and microtubules. Exposure of these cells to colcemid results in a rapid disaggregation of microtubules, and a slow aggregation of the desmin-containing filaments towards the nuclear area with the ultimate formation of a perinuclear ring. In differentiated skeletal or cardiac muscle cells, in addition to its cytoplasmic filamentous distribution, desmin is found intimately associated with the Z lines of sarcomeres. We further show that approx. 50% of the cells in these primary cardiac cultures are unreactive with desmin antibodies. Similarly the majority of the cells in a number of established cell lines from various species grown in tissue culture, are unreactive to desmin antibodies in indirect immunofluorescence, despite the fact that these cells are known to contain cytoplasmic 100Å-filaments. These results indicate that desmin occurs in at least two distinct cytoplasmic distribution in cardiac cells. They also demonstrate the existence of immunological and biochemical differences in the major component of 100Å-filaments between muscle and non-muscle cells as evidenced by the failure of non-muscle cells to react with antibodies to chick smooth muscle desmin.     

The distribution of desmin (100 Å) filaments in primary cultures of embryonic chick cardiac cells

Using antibodies to desmin, the major component of the 100Å-filaments from smooth muscle cells, we studied by indirect immunofluorescence the distribution of this protein in primary cultures of embryonic chick cardiac cells. We show that desmin is a component of cytoplasmic filamentous structures which comprise a network distinct from actin filament bundles and microtubules. Exposure of these cells to colcemid results in a rapid disaggregation of microtubules, and a slow aggregation of the desmin-containing filaments towards the nuclear area with the ultimate formation of a perinuclear ring. In differentiated skeletal or cardiac muscle cells, in addition to its cytoplasmic filamentous distribution, desmin is found intimately associated with the Z lines of sarcomeres. We further show that approx. 50% of the cells in these primary cardiac cultures are unreactive with desmin antibodies. Similarly the majority of the cells in a number of established cell lines from various species grown in tissue culture, are unreactive to desmin antibodies in indirect immunofluorescence, despite the fact that these cells are known to contain cytoplasmic 100Å-filaments. These results indicate that desmin occurs in at least two distinct cytoplasmic distribution in cardiac cells. They also demonstrate the existence of immunological and biochemical differences in the major component of 100Å-filaments between muscle and non-muscle cells as evidenced by the failure of non-muscle cells to react with antibodies to chick smooth muscle desmin.     

Differentiation of vascular pseudointima under normal and disturbed blood flow conditions: Ultrastructural observations in the rat

Summary To study the effect of haemodynamic stress on the morphological differentiation of pseudointima, the ultrastructure of the cells lining normally shaped and aneurysmal polyurethane vascular prostheses implanted into the abdominal aorta of rats was examined. In the normally shaped vascular prostheses the pseudointima was composed of several layers of smooth muscle cells, which varied in differentiation from normal smooth muscle cells to myofibroblasts, and which were lined by a continuous sheet of endothelial cells. In the aneurysmal vascular prostheses, a pseudointima, composed of only layers of smooth muscle cells had developed. Those smooth muscle cells which lined the lumen had a typical morphology: they were polygonal, flat cells of unequal size, with a distinct organelle-free zone, containing myofilaments, at the luminal peripheral cytoplasmic side. The other smooth muscle cells varied in differentiation from normal smooth muscle cells to myofibroblasts. Under severe haemodynamic stresses, such as occur in the aneurysmal vascular prostheses, the regeneration of endothelial cells is impaired and smooth muscle cells undergo morphological changes to form a pseudoendothelial lining.     

Specificity of desmin to avian and mammalian muscle cells.

The extent of invariance and heterogeneity in desmin, the major component of the muscle form of 100 Å filaments, has been investigated in avian and mammalian muscle and nonmuscle cells with two-dimensional gel electrophoresis and indirect immunofluorescence. Desmin from chick, duck and quail, smooth, skeletal and cardiac muscle cells is resolved into two isoelectric variants, α and β, with each possessing the same charge and electrophoretic mobility in all three avian species irrespective of muscle type. Guinea pig and rat muscle desmin resolves into only one variant; it also possesses the same charge and electrophoretic mobility in the two mammalian species, but it is more acidic and slower in electrophoretic mobility than the two avian variants.In immunofluorescence, desmin is localized together with α-actinin along myofibril Z lines. Antibodies to chick smooth muscle desmin, prepared against the protein purified by preparative SDS gel electrophoresis prior to immunization, cross-react with myofibril Z lines in all three avian species. These antibodies do not cross-react with either rat or guinea pig myofibril Z lines. Similarly, they do not cross-react with avian or mammalian nonmuscle cells grown in tissue culture and known to contain cytoplasmic 100 Å filaments.These results demonstrate that desmin is highly conserved within avian muscle cells and within mammalian muscle cells. It is, however, both biochemically and immunologically distinguishable between avian and mammalian muscle cells, and between muscle and nonmuscle cells. We conclude that there are biochemically and immunologically specific forms of desmin for avian and mammalian muscle cells. Furthermore, within a particular vertebrate species, there are at least two separate classes of 100 Å filaments: the muscle class whose major component is desmin, and the nonmuscle class whose major component is distinct from desmin. Taking into consideration the immunological specificity reported by other laboratories for the 100 Å filaments in glial cells, for neurofilaments and for the epidermal 80 Å keratin filaments, we propose that a given vertebrate species contains at least four major distinguishable classes of 100 Å filaments: muscle 100 Å filaments (desmin filaments), glial filaments, neurofilaments and epidermal keratin filaments.     

Trypanosoma cruzi: cross-reactive anti-heart autoantibodies produced during infection in mice

Preimmunization with attenuated Corpus Christi stain Trypanosoma cruzi provides survival to C3H mice and enhances resistance of C57 mice to Brazil strain infection. C3H(He) and C57 B1/6 mice surviving acute infection of T. cruzi are shown to have heart specific autoantibodies through acute and chronic infection. ELISA assays were performed using nondenatured extract of hearts from normal syngeneic mice as target antigen reacted with sera from immunized and/or infected mice. Surviving C3H mice developed a specific anti-heart response as early as Day 21 of infection and this response continued at a high level to Day 300. The response in C57 mice, both immunized-infected and infected only, increased to Day 100 followed by a decline in intensity. The heart specificity of the response in mice was suggested by negligible reaction of sera with smooth muscle preparations and a reduced autoreactivity with skeletal muscle. Laminin, a suggested target of autoimmunity in Chagas' disease, was shown not to be the target of the responses in these mice. Immunoaffinity-purified heart specific antibodies show strong cross-reactivity with parasite antigen and like purified parasite specific antibodies, reacted with heart antigen.     

Topographical difference of cytoskeletal organization in smooth muscle cells of rat duodenum revealed by quick-freezing and deep-etching method

The sarcolemmal domain of rat duodenal smooth muscle cells includes caveolae and associated cytoskeletal or filamentous elements. We have used the quick-freezing, deep-etching method to examine the three dimensional relationships between these components. Replica membranes for separated strips of rat duodenal muscle layers were routinely prepared after extraction soluble proteins from cytoplasm and extracellular matrix. As results, 1) cytoskeletal elements in smooth muscle cells consisted mainly of striated thin filaments; 2) thin filaments were connected with some plasma membranes through filaments associated with the sarcolemma, which formed fine network structures beneath the sarcolemma; 3) many bridging structures between the filaments associated with the sarcolemma and the extracellular matrix were frequently detected in the plasma membrane; and 4) compact filaments associated with the sarcolemma almost disappeared near the caveolae, and only thin filaments were anchored to their neck parts. The special arrangement of the cytoskeletal components, which is probably necessary for the intestinal motility, characterizes the topographical difference of the smooth muscle sarcolemma.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

A pulmonary artery endothelial factor causes unidirectional alignment of smooth muscle cells

Pulmonary artery and aorta endothelial and smooth muscle cells harvested from near term Rambouillet lambs were cultured. To determine whether cultured endothelial cells produce a factor which alters the shape and alignment of smooth muscle cells, rhodamine phalloidin staining of F-actin filaments was carried out, as well as time-lapse cinematography. We were able to demonstrate, with pulmonary artery but not with aortic cells in culture, an endothelial-derived factor which causes the smooth muscle cells to migrate and align in uniform direction. The effect could not be enhanced by making the endothelial cells hypoxic and was similar to that produced by exposing smooth muscle cells to KCl or directly to hypoxia.     

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.     

Occurrence and immunolocalization of plectin in tissues

Various tissues from rat were examined for the occurrence and cellular localization of plectin, a 300,000-dalton polypeptide component present in intermediate filament-enriched cytoskeletons prepared from cultured cells by treatment with nonionic detergent and high salt solution. The extraction of liver, heart, skeletal muscle, tongue, and urinary bladder with 1% Triton/0.6 M KCl yielded insoluble cell residues that contained polypeptides of Mr 300,000 in variable amounts. These high Mr polypeptide species and a few bands of slightly lower Mr (most likely proteolytic breakdown products) were shown to react with antibodies to rat glioma C6 cell plectin using immunoautoradiography and/or immunoprecipitation. By indirect immunofluorescence microscopy using frozen sections (4 micron) of stomach, kidney, small intestine, liver, uterus, urinary bladder, and heart, antigens reacting with antibodies to plectin were found in fibroblast, endothelial, smooth, skeletal, and cardiac muscle, nerve, and epithelial cells of various types. Depending on the cell type, staining was observed either throughout the cytoplasm, or primarily at the periphery of cells, or in both locations. In hepatocytes, besides granular staining at the cell periphery, conspicuous staining of junctions sealing bile canaliculi was seen. In cardiac muscle strong staining was seen at intercalated disks and, as in skeletal muscle, at Z-lines. In cross sections through smooth muscle, most strikingly of urinary bladder, antibodies to plectin specifically decorated regularly spaced, spot-like structures at the cell periphery. By immunoelectron microscopy using the peroxidase technique, antiplectin-reactive material was found along cell junctions of hepatocytes and was particularly enriched at desmosomal plaques and structures associated with their cytoplasmic surfaces. A specific immunoreaction with desmosomes was also evident in sections through tongue. In cardiac muscle, besides Z-lines, intercalated disks were reactive along almost their entire surface, suggesting that plectin was associated with the fascia adherens, desmosomes, and probably gap junctions. In smooth muscle cells, regularly spaced lateral densities probably representing myofilament attachment sites were immunoreactive with plectin antibodies. The results show that plectin is of widespread occurrence with regard to tissues and cell types. Furthermore, immunolocalization by light and electron microscopy at junctional sites of various cell types and at attachment sites of cytoplasmic filaments in epithelial and muscle cells suggests that plectin possibly plays a universal role in the formation of cell junctions and the anchorage of cytoplasmic filaments.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Potassium chloride-insoluble myofilaments in vertebrate smooth muscle cells

Actomyosin was extracted from avian gizzard smooth muscle. The residue was then homogenized and fractionated by differential centrifugation. Fractions of the residue that sedimented at 1 000 g and 13 000 g were examined in negatively stained and sectioned preparations with the electron microscope. The major components of both fractions were 100 Å diameter filaments and fusiform dense bodies. The filaments and dense bodies closely resembled their counterparts in sectioned preparations of unextracted smooth muscle cells from Taenia coli. The insolubility of the 100 Å diameter filaments at high ionic strength and their detailed structure suggest that they are not composed of actin and myosin. Their general features indicate that they correspond to the so-called thick filaments observed in the early studies of vertebrate smooth muscle cells.     

Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland

Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland.