Elias Lazarides

Desmin and vimentin coexist at the periphery of the myofibril Z disc

Two-dimensional gel electrophoresis has revealed that vimentin, the predominant subunit of intermediate filaments in cells of mesenchymal origin, is a component of isolated skeletal myofibrils. It thus coexists in mature muscle fibers with desmin, the major subunit of muscle intermediate filaments. Antisera to desmin and vimentin, shown to be specific for their respective antigens by two-dimensional immunoautoradiography, have been used in immunofluorescence to demonstrate that vimentin has the same distribution as desmin in skeletal muscle. Both desmin and vimentin surround each myofibril Z disc and form honeycomb-like networks within each Z plane of the muscle fiber. This distribution is complementary to that of alpha-actinin within a given Z plane. Desmin and vimentin may thus be involved in maintaining the lateral registration of sarcomeres by transversely linking adjacent myofibrils at their Z discs. This linkage would support and integrate the fiber as a whole, and provide a molecular basis for the cross-striated appearance of skeletal muscle.

Widespread occurrence of avian spectrin in nonerythroid cells

We have prepared an antibody against chicken erythrocyte alpha spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with alpha spectrin in chicken erythrocytes and crossreacts with alpha spectrin in erythrocytes from various mammals. Immunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well.

The synthesis and distribution of desmin and vimentin during myogenesis in vitro

Electrophoretic and autoradiographic analyses of the incorporation of 35S--methionine into newly synthesized proteins during myogenesis reveal that presumptive chicken myoblasts synthesize primarily one intermediate filament protein: vimentin. Desmin synthesis is initiated at the onset of fusion. Synthesis rates of both filament subunits increase during the first three days in culture, relative to the total protein synthesis rate. The observed increase in the rate of desmin synthesis (at least 10 fold) is significantly greater than that observed for vimentin, and is responsible for a net increase in the cellular desmin content relative to vimentin. Both filament subunits continue to be synthesized through at least 20 days in culture. Immunofluorescent staining using desmin- and vimentin-specific antisera supports the conclusion that desmin is synthesized only in fusing or multinucleate cells. These results indicate that the synthesis of the two filament subunits is not coordinately regulated during myogenesis. The distributions of desmin and vimentin in multinucleate chicken myotubes are indistinguishable, as determined by double immunofluorescence techniques. In early myotubes, both proteins are found in an intricate network of free cytoplasmic filaments. Later in myogenesis, several days after the appearance of alpha--actinin-containing Z line striations, both filament proteins become associated with the Z lines of newly assembled myofibrils, with a corresponding decrease in the number of cytoplasmic filaments. This transition corresponds to the time when the alpha--actinin-containing Z lines become aligned laterally. These data suggest that the two intermediate filament systems, desmin and vimentin, have an important role in the lateral organization and registration of myofibrils and that the synthesis of desmin and assembly of desmin-containing intermediate filaments during myogenesis is directly related to these functions. These results also indicate that the Z disc is assembled in at least two distinct steps during myogenesis.

Synemin: a new high molecular weight protein associated with desmin and vimentin filaments in muscle

A 230,000 dalton polypeptide co-purifies through cycles of depolymerization and polymerization with the intermediate filament subunits, desmin and vimentin, from avian smooth muscle. This protein is also present in skeletal muscle and is distinct from myosin and filamin. Double immunofluorescence microscopy of cultured cells, using antisera shown to be specific by immunoautoradiography, has revealed that this protein has the same spatial distribution as desmin and vimentin. During skeletal myogenesis, all three antigens exist initially in multinucleate myotubes as wavy filaments throughout the cytoplasm. Within a week after myoblast fusion, they begin to coalesce at the peripheries of the myofibril Z discs, thereby attaining the distribution observed in mature muscle, a network of interlinked rings within the Z plane. Treatment of cultured myotubes with colcemid causes the filamentous forms of these three proteins to co-aggregate into cytoplasmic bundles, but has little effect on them when they are associated with the Z discs. Extraction of cells with nonionic detergent and high salt leaves cytoskeletons containing desmin, vimentin and the 230,000 dalton polypeptide with immunofluorescent patterns that are indistinguishable from one another. These data suggest that this high molecular weight protein is closely associated with desmin and vimentin filaments in muscle cells; to indicate this, we have named the protein synemin, from the Greek oa uv (with) and v eta mu alpha (filament).

The existence of an insoluble Z disc scaffold in chicken skeletal muscle

Extraction of glycerinated chicken skeletal muscle with 0.6 M potassium iodide leaves a framework of insoluble components within each muscle fiber. This framework is composed primarily of planes of in-register Z discs that have been thickened by the accumulation of material on both sides of each disc during extraction. Membrane vesicles, presumably remnants of the T system, remain surrounding the Z discs. When the framework is sheared in a blender, it is preferentially cleaved between Z planes, resulting in the formation of large sheets of interconnected, closely packed Z discs in a honeycomb-like array. Cleavage occurs in regions formerly occupied by the A bands, which have been weakened by the removal of myosin. The existence and stability of these planar Z disc arrays demonstrate the presence and strength of connections between adjacent myofibrils. SDS-polyacrylamide gel electrophoresis reveals that this framework consists primarily of actin and desmin, with lesser amounts of a few proteins including alpha-actinin, myosin and tropomyosin. Z disc sheets and KI-extracted myofibrils provide a distinct face-on view and side view, respectively, of the Z disc. In indirect immunofluorescence, these two views have revealed that desmin is present at the periphery of each Z disc, forming a network of proteinaceous collars within the Z plane. alpha-Actinin is localized within each disc, giving a face-on fluorescence pattern that is complementary to that of desmin. Actin is present throughout the thickened Z plane, while myosin and tropomyosin exist only in the insoluble residue that coalesces on both faces of each disc. We conclude that desmin, perhaps in conjunction with actin, is responsible for interlinking Z discs of adjacent myofibrils, and may thus serve as a mechanical and structural integrator of muscle fibers. Its hydrophobic nature and coincident distribution with the T system suggest that it may also be responsible for mediating filament-membrane interactions and anchoring the triad to the Z disc. Its collar-like distribution suggests that it may aid in maintaining the structural integrity of the Z disc and the actin filaments inserted into it.

Specificity of desmin to avian and mammalian muscle cells.

Abstract The extent of invariance and heterogeneity in desmin, the major component of the muscle form of 100 A 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 A 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 A 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 A filaments in glial cells, for neurofilaments and for the epidermal 80 A keratin filaments, we propose that a given vertebrate species contains at least four major distinguishable classes of 100 A filaments: muscle 100 A filaments (desmin filaments), glial filaments, neurofilaments and epidermal keratin filaments.

Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy

Intermediate filament structure and distribution were studied by antibody decoration and low-angle shadowing of sonicated chicken erythrocytes and embryonic erythroid cells. Intermediate filaments containing vimentin and synemin form a three-dimensional network in these cells, interlinking the nucleus and plasma membrane. This filament network is spatially segregated from the marginal band of microtubules, indicating that these two systems do not interact directly in the development or maintenance of cell shape. Incubation of sonicated cells with an antiserum specific for vimentin results in uniform decoration of the intermediate filaments; incubation with antisynemin results in decoration of periodically spaced foci. Measurement of the synemin periodicity under a specified set of sample preparation conditions gives average values of 180 nm for adult erythrocytes and 230 nm for 10 day old embryonic erythroid cells, suggesting some fundamental change in the structure of the filaments during erythropoiesis. Registration of these foci in laterally associated filaments, and decoration of bridges between slightly separated filaments, suggest that synemin mediates crosslinking of intermediate filaments through self-interaction.

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.

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

Abstract Using antibodies to desmin, the major component of the 100A-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 100A-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 100A-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.

Phosphorylation of intermediate filament proteins by cAMP-dependent protein kinases

The intermediate filament proteins, desmin and vimentin, are phosphorylated in skeletal muscle cells in vivo. Desmin kinase activities have been purified from mature chicken skeletal muscle and identified as the cAMP-dependent kinases. Chicken skeletal muscle contains two cAMP-dependent protein kinases which are similar to those of other tissues in their subunit composition and chromatographic behavior. The catalytic subunits purified from the two chicken cAMP-dependent kinases phosphorylate both desmin and vimentin in vitro, using cytoskeletal residues prepared from cultured myogenic cells as a substrate. Likewise, the purified catalytic subunits of the rabbit skeletal muscle and bovine heart cAMP-dependent protein kinases phosphorylate desmin and vimentin in vitro. Desmin and vimentin phosphorylation by the rabbit skeletal muscle catalytic subunit is inhibited by the addition of its regulatory subunit. This inhibition is reversed by the presence of cAMP in the reaction mixture. A small fraction of the vimentin phosphorylation is cAmP-independent. Tryptic peptide analysis of desmin phosphorylated in vivo shows two major phosphopeptides. Serine is the phosphorylated amino acid in both peptides. The same two peptides are phosphorylated in vitro by the bovine heart catalytic subunit, but additional peptides are also phosphorylated.