Structural and functional evaluation of branched myofibers lacking intermediate filaments

Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des(-/-)), keratin 19 (K19(-/-)), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca(2+) kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca(2+) kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des(-/-), K19(-/-), or DKO mice and compared them to age-matched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des(-/-) myofibers had more malformations (4.7%) than K19(-/-) (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca(2+) signals showed a decrease in the rate of Ca(2+) uptake (decay rate) into the sarcoplasmic reticulum after Ca(2+) release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca(2+) clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca(2+) reuptake and myofiber branching.     

Sarcolemmal organization in skeletal muscle lacking desmin: evidence for cytokeratins associated with the membrane skeleton at costameres

The sarcolemma of fast-twitch muscle is organized into "costameres," structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain beta-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/- quadriceps, by contrast, most costameric structures are stable. Alternatively, Z line domains may be lost, whereas domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/- muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/- muscle and redistribute with beta-spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.     

Specific interaction of the actin-binding domain of dystrophin with intermediate filaments containing keratin 19

Cytokeratins 8 and 19 concentrate at costameres of striated muscle and copurify with the dystrophin-glycoprotein complex, perhaps through the interaction of the cytokeratins with the actin-binding domain of dystrophin. We overexpressed dystrophin's actin-binding domain (Dys-ABD), K8 and K19, as well as closely related proteins, in COS-7 cells to assess the basis and specificity of their interaction. Dys-ABD alone associated with actin microfilaments. Expressed with K8 and K19, which form filaments, Dys-ABD associated preferentially with the cytokeratins. This interaction was specific, as the homologous ABD of betaI-spectrin failed to interact with K8/K19 filaments, and Dys-ABD did not associate with desmin or K8/K18 filaments. Studies in COS-7 cells and in vitro showed that Dys-ABD binds directly and specifically to K19. Expressed in muscle fibers in vivo, K19 accumulated in the myoplasm in structures that contained dystrophin and spectrin and disrupted the organization of the sarcolemma. K8 incorporated into sarcomeres, with no effect on the sarcolemma. Our results show that dystrophin interacts through its ABD with K19 specifically and are consistent with the idea that cytokeratins associate with dystrophin at the sarcolemma of striated muscle.     

Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibers

Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des-/-) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des-/- muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des-/- and Des+/+. In width measurements of single fibers and bundles, Des-/- soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des-/- and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des-/- soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.     

Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice.

Desmin, the main component of intermediate filaments (IFs) in mature skeletal muscle, forms an interlinking scaffold around myofibrils with connections to the sarcolemma and the nuclear membrane. Desmin is enriched in neuromuscular and myotendinous junctions. Mice lacking the desmin gene develop normally and reproduce. However, postnatally they develop a cardiomyopathy and a dystrophy in highly used muscles. We have investigated whether and how neuromuscular and myotendinous junctions are affected and whether nestin compensates for the lack of desmin in the knock-out (K/O) mice. We show that neither neuromuscular nor myotendinous junctions were markedly affected in the desmin K/O mice. In neuromuscular junctions nestin was present between the postjunctional folds and the subneural nuclei and between the nucleus and the myofibrillar cytoskeleton. In myotendinous junctions nestin was present between myofibrils at the Z-disc level and in longitudinal strands close to and at the junction. Nestin expression at these specialized sites, as well as during myogenesis and myofibrillogenesis, is independent of the presence of desmin. In desmin K/O mice nestin was also found in regenerating myofibers. The presence of nestin at neuromuscular and myotendinous junctions might provide enough strength for preservation and organization of the junctional areas, although desmin is lacking.     

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.     

Utrophin deficiency worsens cardiac contractile dysfunction present in dystrophin-deficient mdx mice

The loss of dystrophin in patients with Duchenne muscular dystrophy (DMD) causes devastating skeletal muscle degeneration and cardiomyopathy. Dystrophin-deficient (mdx) mice have a much milder phenotype, whereas double knockout (DKO) mice lacking both dystrophin and its homolog, utrophin, exhibit the clinical signs observed in DMD patients. We have previously shown that DKO and mdx mice have similar severities of histological features of cardiomyopathy, but no contractile functional measurements of DKO heart have ever been carried out. To investigate whether DKO mice display cardiac dysfunction at the tissue level, contractile response of the myocardium was tested in small, unbranched, ultrathin, right ventricular muscles. Under near physiological conditions, peak isometric active developed tension (F(dev), in mN/mm2) at a stimulation frequency of 4 Hz was depressed in DKO mice (15.3 +/- 3.7, n = 8) compared with mdx mice (24.2 +/- 5.4, n = 7), which in turn were depressed compared with wild-type (WT) control mice (33.2 +/- 4.5, n = 7). This reduced Fdev was also observed at frequencies within the murine physiological range; at 12 Hz, Fdev was (in mN/mm2) 11.4 +/- 1.8 in DKO, 14.5 +/- 4.2 in mdx, and 28.8 +/- 5.4 in WT mice. The depression of Fdev was observed over the entire frequency range of 4-14 Hz and was significant between DKO versus mdx mice, as well as between DKO or mdx mice versus WT mice. Under beta-adrenergic stimulation (1 micromol/l isoproterenol), Fdev in DKO preparations was only (in mN/mm2) 14.7 +/- 5.1 compared with 30.9 +/- 8.9 in mdx and 41.0 +/- 4.9 in WT mice. These data show that cardiac contractile dysfunction of mdx mice is generally worsened in mice also lacking utrophin.     

The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of gamma-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, alpha-actinin, beta-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with gamma-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.     

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) ^1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency ³. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction ⁴. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues ⁵. This later change increases muscle stiffness ⁶. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.     

Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.     

Disruption of muscle architecture and myocardial degeneration in mice lacking desmin

Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.     

Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality

It has been reported previously that keratin 8 (K8)-deficient mice of one strain die from a liver defect at around E12.5, while those of another strain suffer from colorectal hyperplasia. These findings have generated considerable confusion about the function of K8, K18 and K19 that are co-expressed in the mouse blastocyst and internal epithelia. To resolve this issue, we produced mice doubly deficient for K18 and K19 leading to complete loss of keratin filaments in early mouse development. These embryos died at around day E9.5 with 100% penetrance. The absence of keratins caused cytolysis restricted to trophoblast giant cells, followed by haematomas in the trophoblast layer. Up to that stage, embryonic development proceeded unaffected in the absence of keratin filaments. K18/19-deficient mouse embryos die earlier than any other intermediate filament knockouts reported so far, suggesting that keratins, in analogy to their well established role in epidermis, are essential for the integrity of a specialized embryonic epithelium. Our data also offer a rationale to explore the involvement of keratin mutations in early abortions during human pregnancies.     

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.     

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.     

Models of skeletal muscle to explain the increase in passive stiffness in desmin knockout muscle

Absence of desmin in skeletal muscle was found to induce an increase in passive stiffness. The present study aimed at developing rheological models of passive muscle to explain this stiffening. Models were elaborated by using experimental data depicting muscle viscoelastic behaviour. The experimental protocol included stepwise extension tests applied on control and desmin knockout soleus muscles from mice. Linear and non-linear models were composed of elastic and viscous elements. They were constructed with the aim at taking the presence or absence of desmin into account by simulating desmin as an elastic element. Furthermore, associated adaptation of connective tissues in absence of desmin was modelled as an additional elastic element. Differences in passive behaviour induced by absence of desmin were predicted by using a linear model and a non-linear one. The non-linear model was selected because: (1) it is able to predict experimental viscoelastic kinetics accounting for the increase in passive stiffness in muscles lacking desmin, (2) its design is consistent with morphological data, and (3) stiffness characteristics of its elements are in accordance with the literature. Finally, this modelling approach demonstrates that both absence of desmin and adaptation of connective tissue are required to explain the increase in passive stiffness in desmin knockout muscles.     

Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.     

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.