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Vol. 20, Issue 3, 834-845, February 1, 2009
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Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724
Submitted June 23, 2008;
Revised October 14, 2008;
Accepted October 30, 2008
Monitoring Editor: Robert G. Parton
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Giant nebulin molecules span the entire length of the thin filaments, with their N-terminal region interacting with the pointed end of the filaments and their C-terminal region anchored at the Z-discs. Previously, a yeast two-hybrid study reported that the C-terminal region of nebulin interacts with the desmin, illustrating a direct link between sarcomeres and IFs (Bang et al., 2002).
Like other IF proteins, desmin is composed of three major structural domains (for review see Herrmann and Aebi, 2004). At its N-terminal end is a nonconserved, non-
-helical head domain; in the middle is a conserved amphipathic -helical coiled-coiled rod domain that contains three linker regions, and at its C-terminal end is a nonconserved tail domain (Geisler and Weber, 1982). Desmin filaments form a 3D cytoplasmic scaffold linking individual myofibrils laterally at the periphery of the Z-discs connecting the sarcomere to the sarcolemma, T-tubules, mitochondria, and nucleus (Lazarides and Hubbard, 1976; Lazarides, 1978). Desmin is also enriched at intercalated discs/desmosomes, myotendinous junctions, and in Purkinje fibers and is inconsistently detected at the M-line (Tokuyasu et al., 1983; Thornell et al., 1985; O'Neill et al., 2002). Together, the multiple cellular localizations of the desmin filaments are thought to maintain the structural integrity of myocytes and contribute to the force transmission and longitudinal load bearing (e.g., Shah et al., 2004). Desmin –/– mice develop cardiomyopathies characterized by mitochondrial abnormalities, extensive cardiomyocyte death, fibrosis, calcification, and eventual heart failure (Weisleder et al., 2004). Skeletal and cardiac muscle from these desmin –/– mice exhibit misaligned sarcomeres and disintegrated myofibrils, as well as accumulation of mitochondria (Milner et al., 1996; Li et al., 1997). Collectively, these studies highlight the importance of desmin IF in structurally integrating the striated muscle cytoskeleton.
Nebulin is a highly modular protein composed of a unique N-terminal domain, a central region containing 
35 residue actin-binding repeats organized into single modules (M) (M1-8 and M163-185) and into sets of seven-module super-repeats (M9-162) and a C-terminal end region containing a serine-rich linker and SH3 domain (e.g., Labeit and Kolmerer, 1995; Wang et al., 1996). Each single nebulin repeat is predicted to bind an actin monomer with the potential of nebulin binding 200 actin monomers along its length (Pfuhl et al., 1996; Wang et al., 1996). Nebulin represents 2–3% of total myofibrillar proteins in skeletal muscle (Wang and Wright, 1988), whereas it is present at much lower levels in cardiac muscle (Fock and Hinssen, 2002; Kazmierski et al., 2003; Bang et al., 2006). Several studies have indicated that nebulin likely plays an important role in thin filament regulation, together with actin filament capping proteins, to maintain actin filament lengths (for reviews see Fowler et al., 2006; Horowits, 2006; Pappas et al., 2008). In particular, reduction of nebulin via siRNA approaches resulted in longer thin filament lengths in rat cardiac myocytes, whereas knockout of nebulin in mice resulted up to a 15% reduction in the lengths of the skeletal muscle thin filaments (McElhinny et al., 2005; Bang et al., 2006; Witt et al., 2006). The nebulin –/– mice died within 1–3 wk after birth and had defects in contractile activity and in active tension production. Misaligned sarcomeres, myofibril splitting with moderately wider Z-discs, undefined M-lines, absence of H-zones, and accumulation of mitochondria and nemaline bodies were also reported in nebulin –/– skeletal muscles (Bang et al., 2006; Witt et al., 2006). Remarkably, some of the structural abnormalities described in the nebulin –/– mice are similar to those described for desmin –/– mice (Milner et al., 1996; Li et al., 1997).
Single missense mutations in nebulin or desmin can lead to heterogeneous human muscle disorders that typically follow an autosomal dominant pattern of inheritance. Nebulin mutations can result in nemaline myopathy, a disease of the thin filament that is characterized by the presence of nemaline bodies between muscle fibers that contain aberrantly arranged Z-disc and I-band proteins (for review see Wallgren-Pettersson et al., 2007). Missense mutations in desmin can result in dilated and restrictive cardiomyopathies, as well as desmin-related myopathy (DRM), a distinct form of myofibrillar myopathy (Taylor et al., 2007). DRM is characterized by cytoplasmic desmin-positive aggregates and streaming of Z-bands (for review see Goldfarb et al., 2004). Many mutations (>40 thus far) scattered throughout desmin have been found to result in DRM (e.g., Vrabie et al., 2005). Earlier studies proposed that the DRM mutations affected IF network assembly. However, this idea does not explain the pathology observed in many DRM cases. For example, it was found that six DRM-associated desmin rod mutants (A213V, E245D, A360P, N393I, Q389P, and D399Y) formed seemingly normal IFs in in vitro assembly assays (Bar et al., 2005). Likewise, a number of desmin tail mutant proteins integrated into preexisting IF networks in C2C12 mouse skeletal myoblasts and formed extended filamentous arrays in in vitro assembly assays (Bar et al., 2007). These studies suggest that the cellular defects that lead to DRM are complex and involve many cellular processes, many of which are poorly understood.
Here, we sought to understand the role of the interaction of the desmin IF network with the integral sarcomeric component, nebulin. Nebulin M160-164 binds to desmin coil IB with high affinity, and the addition of the DRM-associated E245D mutation in this desmin fragment alters its interaction with nebulin. Expression of the E245D desmin mutant results in reduction in the assembly of endogenous desmin and nebulin at the Z-disc and cytoplasmic aggregate formation, as seen in biopsies of diseased DRM muscle. Expression of the desmin E245D mutant also results in two striking alterations in sarcomeric actin filament structure. First, most sarcomeres contained nonuniform, nonstriated phalloidin staining (i.e., absence of gaps in staining in the H-zone) indicating thin filament elongation. Second, phalloidin staining also revealed the presence of actin filaments with reduced lengths in cardiac myocytes. Hence, we propose that alterations in the desmin–nebulin interaction, and in particular, alterations in nebulin function may contribute to the development of DRM in human muscle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The desmin coil IB region was amplified from human skeletal cDNA (MN_001927) and cloned into pGEX4T-1. The following N-terminal green fluorescent protein (GFP)-desmin constructs were cloned into pEGFP-C1 (Clontech, Mountain View, CA): enhanced GFP (EGFP)-desmin containing residues 2-470, EGFP-head containing residues 1-103, EGFP-coil IB containing residues 151-263, EGFP-coil IIB containing residues 304-412, and EGFP-tail containing residues 411-469. The E245D mutation was introduced into GST-desmin and GST-coil IB using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR products were digested with DpnI, and transformed into XL1-blue super competent Escherichia coli. All constructs were verified by sequencing.
Expression, Purification, and Biotinylation of Recombinant Protein Fragments
GST and 6X His-tagged constructs were transformed into BL21-Codon Plus (DE3)-RIL competent E. coli (Stratagene) for protein production. The bacteria were induced with 1 mM IPTG for 2–3 h at 37°C, and pellets were resuspended in 1% Triton X-100 PBS for GST-fusion proteins or in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 8.0) for His-tagged fusion proteins. The protease inhibitors, 0.5 mM Pefabloc, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 µg/ml aprotinin (Calbiochem, San Diego, CA), were added to all buffers used for protein purification. Samples were sonicated on ice and centrifuged, and the supernatants were incubated on glutathione Sepharose 4B resin (GE Healthcare), or for His-tagged proteins on 50% Ni-NTA slurry in a 15-ml conical tube (Qiagen, Valencia, CA) for 1–2 h at 4°C. The beads with the bound proteins were placed in 10-ml disposable columns and were washed either in 1% Triton X-100 PBS for GST-fusion proteins or in 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8.0, for His-fusion proteins. GST-fusion proteins were eluted with 10 mM glutathione, whereas His-fusion proteins were eluted with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0. Samples were analyzed on SDS-PAGE gels, immediately dialyzed into binding buffer (20 mM HEPES, 80 mM KCl, 2 mM MgCl2, pH 7.4), snap-frozen in liquid nitrogen, and stored at –80°C until use. Recombinant protein fragments were biotinylated at a target ratio of 1:10 wt/wt biotin to protein fragment according to the manufacturer's instructions (Zymed, San Francisco, CA). A Coomassie-stained 12% SDS-PAGE gel of each of the recombinant proteins used in this study is shown (Supplemental Figure S2).
Blot Overlay Assays
For blot overlay analysis, flash-frozen rat psoas and heart muscles were pulverized with a mortar and pestle and solubilized in SDS-sample buffer. Samples were incubated at 70°C for 5 min before being resolved on a 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes (0.2 µm, PerkinElmer Life and Analytical Sciences, Boston, MA) and stained with Ponceau-S. Membrane strips were blocked with 5% milk in binding buffer with 0.05% Tween for 1 h at RT and incubated with 0.5–1.0 µg/ml GST-desmin coil IB or GST alone overnight at 4°C. Strips were washed and incubated with HRP-conjugated GST antibodies (GE Healthcare) diluted 1:8000 in binding buffer for 1 h at RT and then incubated in SuperSignal chemiluminescent substrate (Pierce, Rockford, IL) and exposed to BioMax MR film (Eastman Kodak, Rochester, NY). To detect the mobility of endogenous desmin on the blots, the nitrocellulose membranes were stripped at RT with 0.1 M glycine, pH 2.5, in PBS for 15 min and washed in PBS for 15 min, followed by an incubation in 1 M NaCl in PBS for 15 min. After a 15-min wash in PBS, stripped blots were probed with monoclonal anti-desmin antibodies (DE-U-10: Sigma-Aldrich, St. Louis, MO) at 0.8 µg/ml in 5% milk/0.05% Tween/PBS for 1 h at RT. Primary antibody binding was detected with HRP-conjugated anti-mouse antibodies diluted 1:20,000 (Jackson ImmunoResearch, West Grove, PA).
ELISA Assays
Ninety-six-well microtiter ELISA plates were coated with 100 µl of recombinant His-tagged nebulin fragments (40 nM in Figure 3C or 150 nM in Figure 3, A and B) in 0.1 M carbonate buffer (pH 9.6) overnight at 4°C, following a modified protocol from McElhinny et al. (2001). Wells were blocked for 45 min with 0.2% BSA 0.05% Tween in binding buffer for 1 h at RT, followed by 1-h incubation with biotinylated GST-tagged desmin fragments in binding buffer at the indicated concentrations. After washes with binding buffer, wells were incubated with alkaline phosphatase-conjugated streptavidin (Pierce). After washes with binding buffer, the p-nitrophenyl phosphate (pNPP, Sigma-Aldrich; N9389) substrate (1 mg/ml in 0.1 M glycine, 1 mM MgCl2, 1 mM ZnCl2, pH 10.4) was added to the wells and incubated for 30 min at 37°C. Binding of desmin to nebulin fragments was determined by reading the absorbance at 405 nm. No significant binding was detected when the desmin fragments were tested for binding with GST protein alone. The values shown were obtained by subtracting the values from obtained wells containing binding buffer plus 0.2% BSA and the appropriate biotinylated protein fragment in solution (i.e., nonspecific binding). The estimated dissociation constant (Kd), determined from the saturation curves, was calculated from nonlinear regression curves fitted with a one-site binding equation using Prism software (GraphPad, San Diego, CA). Values shown are the average of triplicate wells ± SD.
Cell Culture and Transfections Protocols
Primary cultures of skeletal myocytes were isolated from pectoralis muscle day-11 chick embryos as previously described (Ojima et al., 1999; McElhinny et al., 2005). Myoblasts were plated at a density of 4.5 x 105 cells per 35-mm culture dish coated with BD matrigel (BD Biosciences, San Jose, CA) following the manufacturer's specifications and were maintained for 96 h in growth medium after isolation. Cardiomyocytes were isolated from rat embryonic day 18 hearts, plated at a density of 5 x 105 cells per 35-mm culture dish, and maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin antibiotics as described previously (Gustafson et al., 1987). Myocytes were transfected with Effectene reagent (Qiagen) 24 h after plating according to the manufacturer's recommendations. Cardiac cultures were incubated for an additional 6 d before fixation.
Immunofluorescence Microscopy and Antibodies
To examine the localization of various proteins, myocytes were stained as previously described (McElhinny et al., 2005). All cells were treated with relaxing buffer (150 mM KCl, 5 mM MgCl2, 10 mM MOPS, 1 mM EGTA, 4 mM ATP, pH 7.4) before fixation in 3% paraformaldehyde. Cells were permeabilized with 0.2% Triton X-100 in PBS and blocked in 2% BSA/1% donkey serum in PBS. Myocytes were stained with rabbit anti-desmin (1:5: Biomeda, Foster City, CA), anti-Tmod1 (10 µg/ml), anti-N-terminal titin Z1-Z2 (1:100), anti-C-terminal nebulin M160-164 (10 µg/ml), and anti-C terminal nebulin M176-181 (1:50; kindly provided by Dr. Siegfried Labeit, Universitatsklinikum Mannheim, Germany) antibodies, and monoclonal anti-myosin F59 (1:10 of culture supernatant: kindly provided by F. Stockdale, Stanford University, Stanford, CA), anti-myomesin (1:100: kindly provided by E. Ehler, King's College London, United Kingdom, and J.-C. Perriard, ETH, Zurich, Switzerland) and anti-sarcomeric -actinin (1:10,000: clone EA-53, Sigma-Aldrich) antibodies. When the GFP-tagged desmin fusion proteins were expressed in fibroblasts (contaminating the primary myocytes cultures), they were not detected with the anti-desmin antibodies, suggesting that this antibody recognizes the endogenous protein only (and not the GFP-tagged recombinant proteins) in transfected myocytes (data not shown). Secondary antibodies purchased from Jackson ImmunoResearch and Invitrogen (Carlsbad, CA) included Alexa Fluor 350–conjugated goat anti-mouse IgG (1:250), and Texas Red–conjugated donkey anti-rabbit IgG (1:600). Texas-Red phalloidin (Sigma-Aldrich) was used to label the actin filaments (1:1500).
Images of myocytes were acquired with a Deltavision deconvolution microscope (Applied Precision, Issaquah, WA) with a 100x objective (1.3 NA) using a CoolSnap HQ charged-couple device camera (Photometrics, Tucson, AZ). The images were processed for presentation with Adobe Photoshop CS (San Jose, CA), and statistical analysis was performed using Microsoft Excel (Microsoft, Redmond, WA), and proFit 6.1.2 software (QuantumSoft, Zurich, Switzerland). Intensity plots were performed on three adjacent sarcomeres of Texas Red-phalloidin–stained myocytes (boxes 6.57 x 1.96 µm) and analyzed with ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD). Sarcomere lengths were determined from -actinin staining by measuring the distance between the peaks using the intensity plot tool in ImageJ. Actin filament lengths determined from Texas-Red phalloidin staining were measured using Softworks software from pointed end to pointed end in two independent experiments. Thin filament lengths as determined by staining for Tmod1 (from pointed end to pointed end) were measured using ImageJ. Both sets of measurements (phalloidin and Tmod1) were divided by 2 to obtain thin filament lengths from barbed to pointed ends (half sarcomeres). The mean and SD of phalloidin measurements were computed using proFit 6.1.2 and by fitting the distribution of individual measurements with a Gaussian cumulative distribution function. The statistical significance of the difference in the mean value of the phalloidin measurements was obtained for GFP-desmin coil IB and GFP-desmin coil IB E245D by performing a nonparametric Kolmogorov-Smirnov test.
To visualize the distributions of individual measurements (see Figure 9B), the histograms of the data were plotted and normalized so that the sum of all bins equaled 1 (i.e., the number of measurements falling in each histogram bin was divided by the total number of measurements). A Gaussian curve with the mean and SD as calculated from the individual measurements was overlaid on each histogram. Only the amplitude of the Gaussian curve was fitted to the binned data. This fit was performed using the Levenberg-Marquardt algorithm in proFit 6.1.2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To confirm the interaction of desmin with nebulin and to begin to narrow down the interacting sites, we performed blot overlay assays on lysates of rat psoas and heart muscle. After transfer to nitrocellulose membranes, lysates were probed with recombinant GST-nebulin M160-170 or GST alone, followed by incubation with HRP-conjugated anti-GST antibodies (Figure 1, lanes 1 and 3). GST-nebulin M160-170 bound to several bands including a protein with the same molecular weight as endogenous desmin (52 kDa, Figure 1, lane 1), as determined by Western blot analysis (Figure 1, lane 2). In addition, GST-nebulin M160-170 also bound with a strong signal to two other myofibrillar proteins, with mobility of 100 and 34 kDa, probably corresponding to -actinin and CapZ, respectively. Similar signals were obtained when rat heart muscle lysates were probed with GST-nebulin M160-170 (data not shown).
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-actinin to mark the Z-discs (Figure 2). Rat cardiac myocytes were used because of our strong interest in understanding the molecular mechanisms contributing to certain forms of cardiomyopathies associated with mutations in desmin. The localization of each of the GFP-desmin fragments analyzed in this study is described (Supplemental Table S2).
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GFP-coil IB was also more easily visualized at the Z-disc than any of the other GFP-desmin fragments when expressed in chick skeletal myocytes (Supplemental Figure S1b). Taken together, the prominent localization of desmin coil IB at the Z-disc in both heart and skeletal muscle is consistent with the interaction of this fragment with the C-terminal region of nebulin M160-170 and suggests that the coil IB region of desmin may directly mediate the interaction of desmin with nebulin at the Z-discs.
Desmin Coil IB Binds with High Affinity to Nebulin M160-164
Because C-terminal nebulin was described in a yeast two-hybrid assay to be important for the desmin–nebulin interaction (Bang et al., 2002), our blot overlay experiments suggested that endogenous desmin interacts with M160-170 (Figure 1), and our expression studies demonstrated that desmin coil IB prominently localizes to the Z-discs (Figure 2d), we hypothesized that this region of desmin contains the major nebulin-binding site. Thus, a His-tagged nebulin M160-170 fragment was immobilized onto a microtiter plate and incubated with biotinylated GST-tagged desmin coil IB. Our results showed that desmin coil IB interacted with nebulin M160-170, but not with -actinin that served as a negative control (Figure 3A). To further define the desmin coil IB binding site within nebulin M160-170, several recombinant His-tagged nebulin fragments within this region were generated and tested for binding to desmin coil IB in ELISAs. We determined that desmin coil IB bound similarly to nebulin M160-164 as to nebulin M160-170, whereas significantly less signal was detected with the other C-terminal nebulin fragments encompassing modules 165-167, 169-171, 171-177, and 182-C terminus (Figure 3B). Note, nebulin M178-M181 was not tested because it could not be cloned; these modules are variably spliced (Millevoi et al., 1998). In conclusion, our results show that desmin coil IB appears to be sufficient for binding to nebulin M160-164, although we cannot rule out the possibility that other binding sites within full-length nebulin and desmin, not represented in these fragments, may contribute to the interaction.
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). We focused our attention on a dominantly inherited heterozygous Glu245Asp (E245D) mutation that causes muscle weakness and cardiomyopathy in humans (Vrabie et al., 2005), but was shown to not interfere with the filament assembly properties of desmin in vitro (Bar et al., 2005).
To estimate a Kd for the interaction of desmin coil IB (and the desmin coil IB E245D mutant) with nebulin M160-M164 (the smallest fragment that we found to bind coil IB), another solid-phase binding assay was utilized. Nebulin M160-M164 (40 nM) was immobilized on a microtiter plate and incubated with increasing concentrations of biotinylated desmin coil IB. Although there was some variability in the Kds measured in each individual experiment, our data consistently showed that the interaction of desmin coil IB E245D resulted in increased avidity (e.g., Bmax which indicates the binding capacity) to nebulin M160-164. For example, in the experiment shown, the interaction of nebulin M160-164 with both wild-type desmin coil IB and mutant desmin coil IB E245D was saturable, with a Kd of 35 nM and Kd of 30 nM, respectively (Figure 3C). However, the estimated Bmax was significantly higher for desmin coil IB E245D mutant compared with wild-type desmin coil IB (Bmax of 0.9 and 3.3, respectively, Figure 3C). Hence, our data show that a single mutation in desmin can alter its interaction with nebulin in vitro.
Expression of the Desmin E245D Mutation Results in Loss of Z-disc Localization and Promoted Cytoplasmic Aggregate Formation
To investigate whether the localization of full-length GFP-desmin or GFP-coil IB was perturbed when the E245D mutation was present, we assessed their distribution in cardiac myocytes. Although GFP alone was never found to be distributed in aggregates, wild-type GFP-desmin and to a lesser extent GFP-coil IB were observed to assemble into intracellular aggregates of varying sizes. Full-length GFP-desmin containing the E245D mutation, however, showed a significant decrease in Z-disc localization with a concomitant increase in its distribution as aggregates, compared with wild-type GFP-desmin (Figure 4A, a and b, and B; compare 62 ± 7% aggregate assembly for GFP-desmin with 88 ± 1% for GFP-desmin E245D). Similar trends were obtained when the distribution patterns of GFP-coil IB containing E245D mutation were observed (Figure 4A, c and d, and C; compare 43 ± 3% aggregate assembly for GFP-coil IB with 65 ± 4% for GFP-coil IB E245D). All together, these data revealed that expression of the E245D mutation in desmin decreased the Z-line distribution of this molecule. Remarkably, the intracellular accumulations observed are strikingly similar to the aggregates observed in cardiac and skeletal muscle biopsies from patients carrying the desmin E245D mutation and also frequently seen in patients suffering from all forms of desmin-related myopathies (e.g., Goldfarb et al., 2004; Vrabie et al., 2005).
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-actinin, myosin, actin, or myomesin in aggregates was detected (e.g., Supplemental Figure S4). The Z-disc localization of endogenous desmin was strikingly decreased in cardiomyocytes expressing the E245D mutation (Figures 5 and 6, A and B). Specifically, quantitative analysis showed that expression of GFP-desmin E245D mutant led to a significant decrease (23 ± 6%) compared with cells expressing wild-type GFP-desmin (36 ± 6%) or GFP alone (41 ± 3%; Figure 6A). Interestingly, cells expressing GFP-coil IB increased the ability of endogenous desmin to assemble at the Z-discs compared with GFP alone (76 ± 5 and 52 ± 10%, respectively), whereas a significant reduction of Z-disc assembly was detected in cells expressing GFP-coil IB E245D (31 ± 4%; Figure 6B). In contrast, the endogenous desmin Z-disc distribution in cells expressing a random desmin mutation (K190A) within GFP-coil IB was not significantly different (data not shown).
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Expression of the Desmin Coil IB E245D Mutation Disrupts the Distribution of Myosin
To examine whether the integrity of other sarcomeric components was affected upon expression of the desmin E245D mutant protein, we stained cells expressing the mutant for the thick filament protein myosin, the M-band protein myomesin, titin, and the major Z-disc protein, -actinin (Figure 7). Our results showed a disruption in the distribution of myosin in cells expressing GFP-coil IB E245D (Figure 7i), suggesting that the desmin mutant influences the distribution of the thick filaments. No obvious perturbations of myomesin, titin, and -actinin were observed in cells expressing GFP-coil IB E245D, compared with cells expressing GFP alone or desmin GFP-coil IB (Figure 7, b–d, f–h, and j–l). See Supplemental Figure S1 for the corresponding images of the assembly of the desmin GFP-tagged fragments.
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-actinin antibodies to assess Z-disc structure and spacing (Figure 8). The majority of cells expressing GFP alone or desmin GFP-coil IB contained well organized actin filaments with uniform staining along the lengths of the filaments with bright distinct staining at the Z-line and the absence of staining at the M-line (Figure 8, b and e). This staining pattern indicated uniformity of thin filament lengths at both the barbed and pointed ends. Interestingly, actin filament morphology was significantly altered in cells expressing the GFP-coil IB E245D. Notably, the phalloidin staining at the Z-discs in these mutant cells was often irregular (nonuniform) and broader (see pixel intensity plots in Figure 9B). Additionally, the phalloidin staining at the pointed ends of the filaments was often not well defined, and in many instances gaps at the M-line were not visible (Figures 8h and 9A). These observations suggest thin filament misalignment and/or alterations in filament lengths at both ends of the filaments (Figure 8, compare b and e to h). Remarkably, expression of GFP-coil IB E245D appeared to have no significant effect on the localization of the major Z-disc protein -actinin, as well as Z-disc spacing (i.e., sarcomere lengths) as evidenced by a striated mature staining pattern for -actinin in the identical myofibrils that displayed altered phalloidin staining (Figure 8, compare c and f to i). Similar alterations (i.e., lack of gaps in phalloidin staining at the M- line) in actin filament architecture were observed in nearly all myofibrils in chick skeletal myocytes expressing GFP-coil IB E245D (Supplemental Figure S6).
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13 and 18% of the myofibrils, respectively, showed nonstriated phalloidin staining. However, in cells expressing GFP-coil IB E245D, 60% of the myofibrils displayed nonstriated phalloidin staining (Figure 9A). Similarly, even though 49% of the myofibrils in cells expressing wild-type GFP-desmin displayed nonstriated phalloidin staining, expression of GFP-desmin E245D resulted in 87% of myofibrils lacking distinct gaps at the M-lines (Figure 9A, Supplemental Figure S5). Because the sarcomere lengths (distance between Z-discs) as assessed by staining for -actinin were not altered (i.e., contracted; Figure 8), the presence of nonstriated phalloidin staining suggests that the thin filaments elongated from their pointed ends in cells expressing the E245D mutation and to a lesser extent in cells overexpressing wild-type full-length desmin. We measured phalloidin staining (actin filament lengths) in individual sarcomeres containing discernable gaps at the M-line. This analysis revealed that the actin filaments that could be measured (from pointed end to pointed end) in cells expressing GFP-coil IB E245D were significantly shorter (1.38 ± 0.12 µm) than those found in myocytes expressing GFP (1.58 ± 0.13 µm), or GFP-coil IB (1.56 ± 0.11 µm). We also stained cells with anti-Tmod1 antibodies to mark the pointed ends of the actin filaments; this analysis also demonstrated a significant decrease in actin filament lengths in cells expressing desmin coil IB E245D (Supplemental Table 3). Lastly, the actin filament measurements obtained were analyzed as histograms with curves showing a Gaussian fit to the data, and by performing a two-sample Kolmogorov-Smirnov test (Figure 9C, Supplemental Figure S7). These analyses showed that the distribution of actin filament lengths in cells expressing GFP-coil IB E245D was shifted to the left, indicating reduced thin filament length distributions in cells with the desmin mutant compared with cells expressing desmin GFP-coil IB. Taken together, our data, reveal a novel function for the desmin IFs in regulating actin filament morphology and lengths and suggest that alterations in the desmin–nebulin interaction may contribute to certain DRM disease pathologies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Biochemical support for this became available when it was shown that the giant actin filament–binding protein, nebulin, interacts with desmin at the periphery of the Z-discs (Bang et al., 2002). Nevertheless, the functional significance of the desmin–nebulin interaction in live myocytes and the potential role of this interaction in the context of muscle disease was previously unexplored. In this study, we established a functional link between desmin and nebulin. Our results revealed that expression of a disease-associated point mutation at amino acid 245 (E245D) within the coil-IB region of desmin resulted in disruption of the endogenous desmin and nebulin Z-disc localization and promoted cytoplasmic aggregate formation, mirroring a major histological feature found in human DRM muscle biopsies. Furthermore, our data demonstrated that expression of the desmin E245D mutation dramatically altered actin filament lengths in myocytes. Our findings support a model in which the functional properties of nebulin associated with thin filament length regulation and/or maintenance appear to be, at least in part, modulated by its interaction with desmin. Thus, our data give novel insights into potential molecular consequences underlying DRMs.
Our targeting experiments showed that of all the GFP desmin regions tested, the coil IB region of desmin was consistently the most efficient at localizing to the Z-discs in cardiac and skeletal myocytes, in agreement with its interaction with C-terminal nebulin (Figure 2, Supplemental Figure S1). Because of this result as well as our observation that overexpression of the full-length molecule itself alters thin filament lengths and result in the accumulation of cytoplasmic aggregates, we utilized the wild-type coil IB fragment (which displays little, or no, detectable cellular perturbations) in most of our studies to focus on the interaction of desmin at the Z-disc. A possible explanation for the observed cellular effects on full-length desmin overexpression is that it likely altered the stoichiometric ratios of desmin to nebulin and/or it may have influenced interactions with other desmin-binding partners within myocytes. Interestingly, expression of the wild-type coil IB fragment alone was found to "recruit" endogenous desmin to the Z-disc, suggesting that it has the ability to self-associate with the full-length molecule. Additionally, we observed clear M-line targeting of both the coil IIB and tail regions of desmin. This raises the exciting possibility that desmin is indeed a component of the transverse M-line associated filaments connecting adjacent myofibrils observed in electron micrographs and rarely visualized in confocal studies (Price, 1984; O'Neill et al., 2002); these filaments are thought to contribute to the transverse stability of myofibrils during muscle contraction-relaxation cycles (e.g., Wang and Ramirez-Mitchell, 1983). Our data also suggest that the coil IIB and tail regions of desmin can target efficiently to the M-line in cultured cells because they are missing other regions of full-length desmin that contain interacting sites for non-M-line components that can mostly outcompete for M-line assembly in vivo. This interpretation is consistent with the near absence of GFP-desmin staining at the M-line in cultured myocytes. Based on the findings that the M-band protein EH-myomesin (skelemin), the major embryonic vertebrate heart isoform of myomesin, copurified with desmin and containing two "intermediate filament binding motifs," it has been proposed that it may bind to desmin (Price, 1984; Steiner et al., 1999; Agarkova et al., 2000). The significance of the M-line localization of desmin and identification of its M-line interacting partner(s) remain to be fully elucidated.
Our biochemical evidence revealed that the coil IB region of desmin is important for its interaction with the C-terminal end of nebulin, because desmin coil IB binds with high affinity to nebulin M160-164 with a dissociation constant of Kd 35 nM (Figure 3C). Notably, the affinity of desmin coil-IB and nebulin M160-164 is similar to that reported for other nebulin-protein interactions (e.g., Tmod1 with nebulin, CapZ with nebulin: McElhinny et al., 2005; Pappas et al., 2008). The dissociation constants were not significantly different between wild-type coil IB versus mutant coil IB E245D in ELISAs. However, the addition of the desmin coil IB E245D mutation reproducibly increased its binding capacity for nebulin (the Bmax was >4-fold higher for the mutant: Figure 3C). In agreement with these findings, a competition assay revealed that the coil IB E245D mutant was a more efficient competitor than nonmutated coil IB for the binding of nonmutated coil IB to nebulin M160-164 (the IC50 was 13-fold lower for the mutant: data not shown). One possibility is that the increase in binding capacity of the mutant may alter its binding affinities to other sarcomeric components. Together, these experiments show that both wild-type and mutant desmin coil-IB bind with high affinity to nebulin M160-164 in vitro. Interestingly with respect to our data, based on binding data, Pappas et al., (2008) showed that nebulin modules M160-164 must be present at the edge of the Z-disc and not projecting out from the Z-disc as previously reported. Thus, we propose a model in which desmin maintains the alignment of the myofibrils by directly binding to a segment of nebulin M160-164 facing the periphery of the Z-disc.
Here, we report that overexpression of full-length desmin itself or the coil IB region of desmin containing the E245D mutation results in a marked removal of endogenous desmin from the Z-disc with a concomitant increase in endogenous desmin in intracellular aggregates containing the mutant proteins (Figures 4 and 5). These aggregates appeared similar to those reported in muscle biopsies taken from the patients with the desmin E245D mutation. These findings suggest that our approach of overexpressing the desmin E245D mutation in a model of primary cultures of myocytes is valid, as it resembles the genetic background found in humans, because this particular desmin mutation is heterozygous with an autosomal dominant pattern of inheritance (Vrabie et al., 2005). Interestingly, we also found that expression of the desmin E245D mutation resulted in striking alterations in actin filament organization and lengths as evidenced by 1) nonuniformity in phalloidin staining at the Z-disc, suggesting disorganization and/or filament elongation at their barbed ends; 2) significant increase in nonstriated (i.e., no visible gaps/absence of H-zones) phalloidin staining (without a decrease in sarcomere lengths), suggesting thin filament elongation from their pointed ends; and 3) thin filament shortening in cardiac sarcomeres that contained H-zones in phalloidin staining (Figures 8 and 9 and Supplemental Figure S6). Intriguingly, the alterations in thin filament lengths observed from expression of the E245D desmin mutant are reminiscent of the changes in actin filament lengths observed in nebulin –/– mouse models and in nebulin knockdown studies. For example, in nebulin –/– mouse models, thin filament lengths were significantly shorter (Bang et al., 2006; Witt et al., 2006), whereas nebulin knockdown by siRNA led to both nonstriated (longer) and shorter actin filaments (McElhinny et al., 2005; Pappas et al., 2008). These data, together with the fact that no major changes in the distribution of most (but not all, see below) sarcomeric components were observed as a result of expressing the desmin E245D mutant (Figure 7), are consistent with our prediction that nebulin's actin filament length regulation functions are compromised in cells expressing the desmin E245D. This adds a new functional role of desmin IFs in regulating (at least some of) nebulin's length-regulating properties.
The significance of our findings is particularly noteworthy because perturbations in actin organization and/or lengths would be expected to directly influence the relative range of lengths over which the sarcomeres can generate force effectively. Interestingly, desmin does not require nebulin to localize to the Z-disc as observed in nebulin –/– mice (Bang et al., 2006). However the interaction appears to be important for force transmission, based on the observations reported in nebulin –/– models in which the missing linkage of desmin to the Z-disc to nebulin results in lowered stress generation in the nebulin –/– mice (Bang et al., 2006; Witt et al., 2006). Thus, an interesting question arises as what is the mechanical relationship between desmin and nebulin and how nebulin might aid in transmission of force from the thin filament to the intermediate filament system via its association with desmin at the Z-discs. Consistent with this idea, the observed perturbations in the organization of myosin upon expression of DRM-associated desmin E245D mutation could be influenced by suboptimal interactions with actin filaments of altered lengths (Figure 7i). Another interpretation is that the observed changes in actin filament morphology may result from a nebulin-based misregulation of actin filament–capping protein dynamics (Tmod1 and/or CapZ) at the ends of the thin filaments. A conundrum is that although there appears to be much lower levels of nebulin in cardiac myofibrils, in comparison to skeletal myofibrils (e.g., Kazmierski et al., 2003; Bang et al., 2006), we observed similar thin filament perturbations as a result of the expression of desmin E245D mutant. We, as well as others, speculate that many sarcomeric components contribute to the regulation of thin filament lengths, which may also be affected by the expression of the desmin E245D mutant. This prediction is supported by the fact that invertebrate organisms such as Caenorhabditis elegans and Drosophila (do not have nebulin or desmin orthologues), yet still have uniform thin filament lengths. It is clear that more studies will be required to resolve how the levels of nebulin influence its thin filament length–regulating properties.
In conclusion, our results provide new evidence that the desmin–nebulin interaction plays an important role in muscle physiology and that variations in actin filament lengths may directly contribute to pathogenic molecular consequences underlying certain desmin-related myopathies.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: College of Veterinary Medicine, Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843. 
Present name and address: Syerra N. Lea, Midwestern University, Glendale, AZ 85308.
Address correspondence to: Carol C. Gregorio (gregorio{at}u.arizona.edu)
Abbreviations used: IF, intermediate filaments; DRM, desmin-related myopathies.
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), whereas desmin coil IB E245D mutant fragment binds with a Kd of 
). A molar ratio of 1:1 was used in A and B.
