Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.     

Visualization of myosin in living cells

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.     

Binding and distribution of fluorescently labeled filamin in permeabilized and living cells

This study reports the first development of a fluorescently labeled filamin. Smooth muscle filamin was labeled with fluorescent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bound to the Z bands of isolated cross-striated myofibrils and to the Z bands and intercalated discs in both permeabilized embryonic cardiac myocytes and in frozen sections of adult rat ventricle. In permeabilized embryonic chick myotubes, filamin bound to early myotubes but was absent at later stages. In living embryonic chick myotubes, the fluorescently labeled filamin was incorporated into the Z bands of myofibrils during early and late stages of development but was absent during an intermediate stage. In living cardiac myocytes, filamin-IAR was incorporated into nascent as well as fully formed sarcomeres throughout development. In permeabilized nonmuscle cells, labeled filamin bound to attachment plaques and foci of polygonal networks and to the dense bodies in stress fibers. The periodic bands of filamin in stress fibers had a longer spacing in fibroblasts than in epithelial cells. When injected into living cells, filamin was readily incorporated into stress fibers in a striated pattern. The fluorescent filamin bands were broader in injected cells, however, than they were in permeabilized cells. We have interpreted these results from living and permeabilized cells to mean that native filamin is distributed along the full length of the actin filaments in the stress fibers, with a higher concentration present in the dense bodies. A sarcomeric model is presented indicating the position of filamin with respect to other proteins in the stress fiber.     

Unusual organization of desmin intermediate filaments in muscular dysgenesis and TTX-treated myotubes

Cytoskeletal intermediate filaments were studied in muscular dysgenesis (mdg) and tetrodotoxin-treated inactive mouse embryo muscle cultures during myofibrillogenesis. Both muscular dysgenesis and tetrodotoxin-treated muscles are characterized in vitro by a total lack of contractile activity and an abnormal development of myofibrils. We studied the organization of the microtubule and intermediate filament networks with immunofluorescence, using anti-tubulin, anti-vimentin, and anti-desmin antibodies during normal and mdg/mdg myogenesis in vitro. Mdg/mdg myotubes present a heterogeneous microtubule network with scattered areas of decreased microtubule density. At the myoblast stage, cells expressed both vimentin and desmin. After fusion only desmin expression is revealed. In mutant myotubes the desmin network remains in a diffuse position and does not reorganize itself transversely, as it does during normal myogenesis. The absence of a mature organization of the desmin network in mdg/mdg myotubes is accompanied by a lack of organization of myofibrils. The role of muscle activity in the organization of myofibrils and desmin filaments was tested in two ways: (i) mdg/mdg myotubes were rendered active by coculturing with normal spinal cord cells, and (ii) normal myotubes were treated with tetrodotoxin (TTX) to suppress contractions. Mdg/mdg innervated myotubes showed cross-striated myofibrils, whereas desmin filaments remained diffuse. TTX-treated myotubes possessed disorganized myofibrils and a very unusual pattern of distribution of desmin: intensively stained desmin aggregates were superimposed upon the diffuse network. We conclude, on the basis of these results, that myofibrillar organization does not directly involve intermediate filaments but does need contractile activity.     

Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis

Immunofluorescence microscopy has been used to follow the rearrangement of intermediate-sized filaments during mitosis in rat kangaroo PtK2 cells. These epithelial cells express two different intermediate filament systems: the keratin-related tonofilament-like arrays typical of epithelial cells, and the vimentin-type filaments characteristic of mesenchymal cells in vivo, and of many established cell lines. The two filament systems do not appear to depolymerize extensively during mitosis, but show differences in their organization and display which may indicate different functions. The most striking rearrangements have been seen with the vimentin filaments, and in particular in prometaphase a transient cage-like structure of vimentin fibers surrounding the developing spindle is formed. In metaphase, this cage disappears, and vimentin fibers are found in an elliptical band surrounding the chromosomes and the interzone. In telophase, these bands separate, usually breaking first on the side closest to where the cleavage furrow has started to form. Double label experiments with tubulin and vimentin antibodies have indicated that the microtubules and the chromosomes are contained within the thick crescents of vimentin filaments and suggest that the vimentin intermediate filaments may be involved in the orientation of the spindle and/or the chromosomes during mitosis. In contrast, extensive arrays of cytokeratin filaments are present throughout mitosis on the substrate-attached side of the cell and also in other cellular areas, although they are usually not present in the spindle region. Thus the cytokeratin filaments probably continue to play a cytoskeletal role during mitosis and may be responsible for the flat shape that certain epithelial cells such as PtK2 cells continue to maintain during mitosis.     

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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 α-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 a-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.     

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.     

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.     

Site-specific antibodies block kinase A phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes.

Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP-dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino-terminal headpiece domains; in chicken smooth muscle desmin the most kinase A-reactive residues are ser-29 and ser-35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti-peptide antibodies directed against residues 26-36. The antibodies, which we call anti-D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26-36. Pre-incubation of desmin with affinity purified anti-D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75-80%. When antibody-treated IFs are subjected to phosphorylation, no filament break-down is observed after 3 hours. Thus anti-D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti-D26 at the mononucleated, myoblast stage. By 24 h post-injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation-induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

Interaction of Theiler’s Virus with Intermediate Filaments of Infected Cells

Theiler’s murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler’s virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.     

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.     

Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and promotes apoptosis

Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

Assembly of carboxy-terminally deleted desmin in vimentin-free cells.

Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely.