Bruce L. Granger

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.

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.

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.

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.