Desmuslin, an that interacts with ␣- and

Yuji Mizuno*, Terri G. Thompson*, Jeffrey R. Guyon*, Hart G. W. Lidov*, Melissa Brosius*, Michihiro Imamura†, Eijiro Ozawa†, Simon C. Watkins‡, and Louis M. Kunkel*§

*Howard Hughes Medical Institute͞Division of Genetics, Children’s Hospital and Harvard Medical School, Boston, MA 02115; †National Institute of Neuroscience, National Center for Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan; and ‡Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15261

Contributed by Louis M. Kunkel, March 28, 2001 Dystrobrevin is a component of the -associated protein (19), the rabbit 94-kDa protein (A0) (20), and ␤-dystrobrevin complex and has been shown to interact directly with dystrophin, (21). ␣-Dystrobrevin 1 has a unique C-terminal region with ␣1-, and the complex. The precise role of multiple sites for tyrosine phosphorylation and is highly ex- ␣-dystrobrevin in has not yet been determined. To pressed in muscle and brain. This protein has two predicted study ␣-dystrobrevin’s function in skeletal muscle, we used the ␣-helical coiled-coil motifs and has been shown to interact yeast two-hybrid approach to look for interacting . Three directly with ␣1-syntrophin (16, 17) and dystrophin (12). The overlapping clones were identified that encoded an intermediate ␣-dystrobrevin 2 splice form is slightly different in that it lacks filament protein we subsequently named desmuslin (DMN). Se- the unique C-terminal region and thus would not be phosphor- quence analysis revealed that DMN has a short N-terminal domain, ylated. ␣-Dystrobrevin 3 has an alternatively spliced 3Ј end that a conserved rod domain, and a long C-terminal domain, all common is more truncated than that of ␣-dystrobrevin 2. ␣-Dystrobrevin features of type 6 intermediate filament proteins. A positive 4 and 5 have a different 5Ј start site relative to variants 1–3. interaction between DMN and ␣-dystrobrevin was confirmed with To better understand the role of ␣-dystrobrevin in skeletal an in vitro coimmunoprecipitation assay. By Northern blot analysis, muscle, we looked for interacting proteins by using the yeast we find that DMN is expressed mainly in heart and skeletal muscle, two-hybrid technique. We isolated three overlapping clones and although there is some expression in brain. Western blotting confirmed their interaction with dystrobrevin with in vitro co- detected a 160-kDa protein in heart and skeletal muscle. Immuno- (CoIP). A full-length cDNA clone of the fluorescent microscopy localizes DMN in a stripe-like pattern in interacting protein was isolated. This protein, desmuslin (DMN), longitudinal sections and in a mosaic pattern in cross sections of colocalizes to the Z-lines with desmin. Our results suggest that skeletal muscle. Electron microscopic analysis shows DMN colocal- DMN forms a linkage between desmin and ized with desmin at the Z-lines. Subsequent coimmunoprecipita- and therefore provides an important structural support in tion experiments confirmed an interaction with desmin. Our find- muscle. ings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through ) and may play Materials and Methods an important role in maintaining integrity. Yeast Two-Hybrid Library Screening. ␣-Dystrobrevin cDNA (nucle- otides 7–1617) was inserted downstream of the Gal-4 DNA- he severe muscle wasting disorder, Duchenne muscular binding domain in the pGBT9 bait vector. A yeast two-hybrid Tdystrophy, is caused by abnormalities in the dystrophin cDNA library derived from skeletal muscle was screened (1). The dystrophin protein is expressed in heart and skeletal for interacting proteins with the use of the Matchmaker two- muscle, where it is part of the dystrophin-associated protein hybrid system as described by the distributor (CLONTECH). complex. Dystrophin’s N-terminal domain binds to , In brief, transformation mixtures were spread on synthetic whereas the WW domain and the total cysteine-rich domain bind dropout͞ϪHis͞ϪLeu͞ϪTrp͞ϩ3-amino-1,2,4-triazole plates to ␤- (2), a component of the dystroglycan subcom- and incubated at 30°C until colonies appeared (6 days). Colonies plex. This subcomplex links to , a major component of the able to grow on minimal plates were screened for ␤-galactosidase basal membrane, thereby forming the linkage between an intra- activity with the use of a filter-lift assay as described by the cellular protein, actin, and the extracellular matrix. manufacturer. Yeast DNA isolated from colonies positive for A second subcomplex of the dystrophin-associated protein ␤-galactosidase activity was used to electroporate Escherichia complex includes four transmembrane proteins (␣-, ␤-, ␥-, and coli to recover the interacting cDNA. The sequence of the ␦-sarcoglycan) (3). Each has been shown to be involved in interacting cDNA was analyzed on an ABI 373 or 377 automated different forms of limb-girdle muscular dystrophy (LGMD 2D, sequencer with the use of fluorescent dye terminator chemistry 2E, 2C, and 2F) (4–8). ␣-Sarcoglycan is a type 1 transmembrane (Applied Biosystems). protein and is expressed in heart and skeletal muscle. ␤-, ␥-, and ␦- are type 2 transmembrane proteins containing a Phage cDNA Library Screening. A ␭gt 11 human skeletal muscle cluster of cysteine residues in their extracellular domains. These library (CLONTECH) was screened with a 152-bp hybridization four proteins form the sarcoglycan complex, which is thought to probe homologous to DMN’s 5Ј region (Fig. 2A, nucleotides Ϫ23 be involved in some type of signaling pathway (9). A third subcomplex of the dystrophin-associated protein complex involves ␣-dystrobrevin (10–12) and the Abbreviations: CoIP, coimmunoprecipitation; DMN, desmuslin; IF, intermediate filament. (␣1, ␤1, and ␤2) (13–15). These intracellular proteins directly Data deposition: The sequence reported in this paper has been deposited in the GenBank bind to dystrophin (16, 17). In addition, the N-terminal region of database (accession no. AF359284). ␣-dystrobrevin associates with the sarcoglycan complex (18). §To whom reprint requests should be addressed at: Howard Hughes Medical Institute, Division of Genetics, Enders 570, Children’s Hospital, 300 Longwood Avenue, Boston, MA There are at least five different forms of ␣-dystrobrevin gener- ␣ 02115. E-mail: [email protected]. ated by . The largest splice variant -dystro- The publication costs of this article were defrayed in part by page charge payment. This brevin 1 shows to the cysteine-rich and article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. C-terminal domains of dystrophin, the Torpedo 87-kDa protein §1734 solely to indicate this fact.

6156–6161 ͉ PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153298 Downloaded by guest on September 26, 2021 of the subcloned cDNAs also was expressed from the pFHR2 vector along with the rest of the protein. Proteins expressed from these vectors were termed FLAG 4C, FLAG 5D-1, or FLAG 5D-2 (Fig. 1B). We also made several truncated versions of DMN to identify the subregion that interacted with ␣-dystrobrevin. Constructs were prepared by PCR amplification, using full-length DMN as the template. The DMN PCR product covered the sequence from Ϫ3 to 1011 and was subcloned into pFHR2 to express FLAG 1A-1B-2A-2B (amino acids Ϫ1 to 337). Constructs 1A-1B-2A (amino acids Ϫ1 to 225), 2A-2B (amino acids 161– 337), 1B-2A (amino acids 62–225), C-terminal-1 (amino acids 336–933), and C-terminal-2 (amino acids 932-1253) were cloned in a similar fashion. Clone 4C inserted into pFHR2 was used to express FLAG 1A-1B (amino acids Ϫ34 to 153). Desmin was cloned by PCR amplification from total cellular RNA isolated from human skeletal muscle (CLONTECH). The Fig. 1. CoIP analysis of DMN clones with dystrobrevin. (A) Alignment of PCR product was subcloned into pMGT1 and sequenced. KIAA0353 (22) and DMN clones 4C, 5D, 5D-1, and 5D-2. IF designates a region with an intermediate filament signature. (B) When simultaneously expressed, Antibodies. Synthetic peptides corresponding to DMN-1 amino FLAG 5D-1 coimmunoprecipitates dystrobrevin (compare lane 5 to lane 4). FLAG 5D-2 and FLAG 4C do not coimmunoprecipitate dystrobrevin (lanes 6 acids 232–250 (C-QEAEALRREALGLEQLRAR) and and 7). Our controls show that the FLAG antibody specifically precipitates DMN-2 amino acids 1038–1053 (C-SLSRQRSPAPGSPDEE) proteins with the FLAG epitope (lanes 1–3) and precipitates background levels (Fig. 2A) were generated and used to inject New Zealand White of dystrobrevin (lane 4). * indicates 35S-labeled dystrobrevin. ϩ͞Ϫ indicates rabbits (Research Genetics, Huntsville, AL). The amino- the presence or absence of a particular construct. terminal cysteine is not part of the DMN sequence, but was added for subsequent affinity purification with the use of a SulfoLink coupling gel (Pierce). Anti-FLAG mAb was pur- to 129). The probe was labeled with the use of a Random Primer chased from Sigma. DNA Labeling System (GIBCO͞BRL) in the presence of 5 ␮Ci of [␣-32P]dCTP. Filters were prehybridized in hybridization In Vitro Transcription͞Translation and CoIP. Proteins encoding the buffer [5ϫ SSC (0.75 M NaCl͞0.075 M sodium citrate)͞50 mM various constructs were labeled with [35S]methionine with a TNT sodium phosphate (pH 7.4)͞1ϫ Denhardt’s solution (0.02% Quick Coupled Transcription͞Translation System (Promega). Ficoll [type 400]͞0.02% polyvinylpyrrolidone͞0.02% BSA)] for Depending on the experiment, the proteins were expressed 5.5 h at 65°C. The denatured probe (8.9 ϫ 105 cpm͞ml) was either individually or simultaneously. Five microliters of protein added to the hybridization buffer, and the filters were hybridized lysate was added to 25 ␮l of CoIP buffer [150 mM NaCl͞50 mM for 18 h at 65°C. Filters were washed in 2ϫ SSC and 0.1% SDS Tris⅐HCl (pH 7.4)͞1% Nonidet P-40͞Protease Inhibitor Mixture for 30 min at 55°C with several changes of washing solution. Tablet (Roche)͞50 ml] and mixed for2hat4°C. Subsequently, Filters were wrapped in Saran wrap and exposed to x-ray film at 2 ␮l of anti-FLAG mAb (Sigma) and 18 ␮l of the CoIP buffer Ϫ80°C for 18 h. DNA from positive clones was purified with a were added, and the mixture was then reincubated for3hat4°C. Qiagen (Chatsworth, CA) Lambda Midi Kit. The identity of the After this step, 50 ␮l of suspended protein G-Sepharose (Sigma) cDNA as overlapping with the original clone was verified by was added and shaken at 4°C overnight. The next day, the sequencing. mixture was centrifuged at 1,000 ϫ g for 1 min, and the pellet was washed three times with CoIP buffer and then resuspended in 2ϫ Northern Blot Analysis. A 219-bp hybridization probe against Tris-glycine sample buffer (Novex) with 50 mM DTT. The DMN’s 3Ј region (Fig. 2A, nucleotides 1268–1486) was prepared samples were heated to 85°C for 2 min and separated by by PCR amplification. This probe was then labeled with the use electrophoresis on 10% Tris-glycine acrylamide gels (Novex, San of a Random Primer DNA Labeling System (GIBCO͞BRL) and Diego). Proteins were visualized by exposing the gels to a used to hybridize to a human multiple-tissue RNA blot (CLON- phosphor plate and scanning with a PhosphorImager (Molecular TECH) according to the manufacturer’s protocol. DMN mRNA Dynamics). was visualized by autoradiography. A second hybridization Ϫ

probe, the 152-bp DMN probe (Fig. 2A, nucleotides 23 to 129) Immunoblot Analysis. Human protein medleys (CLONTECH) used for screening the phage cDNA library, was prepared were separated by electrophoresis on 4–20% acrylamide gels, similarly and used to probe the blot. and the proteins were transferred to a nitrocellulose membrane in a transfer buffer (48 mM Tris͞39 mM glycine͞13 mM Constructs. Truncated forms of ␣-dystrobrevin were prepared by SDS͞20% methanol) at 15 V for 20 min with a Trans-Blot PCR amplification: (i) 1–16 (nucleotides 16–1617), (ii) SemiDry apparatus (Bio-Rad). The membrane was blocked with exons 1–14 (nucleotides 16–1386), (iii) exons 8–16 (long) (nu- blocking buffer (1ϫ PBS͞0.1% Tween 20͞5% nonfat milk) cleotides 904-1617), (iv) exons 8–16 (short) (nucleotides 973- overnight at 4°C. The membrane was incubated with anti- 1617), and (v) exons 10–16 (nucleotides 1033–1617). The PCR DMN-1 antibody diluted in blocking buffer for2hatroom products were subcloned into the pMGT1 T7 expression vector. temperature, washed with 1ϫ PBS͞0.1% Tween 20, and then DMN cDNA from clones 4C and 5D was excised from the incubated for1hwithhorseradish peroxidase-conjugated don- pGAD10 library vector with the use of restriction enzymes and key anti-rabbit IgG (H ϩ L) secondary antibody (Jackson was subcloned into pFHR2. The 5D sequence was cloned in two ImmunoResearch). The membrane was washed in 1ϫ PBS͞0.1% pieces by cleaving the clone with EcoRI [see position 434 in Tween 20, and the horseradish peroxidase-conjugated protein KIAA0353 (22)] (Fig. 1A). The pFHR2 vector coexpresses the was detected by chemiluminescence. FLAG epitope on the N-terminal of the subcloned protein. Because of its location downstream of the FLAG sequence and Immunofluorescent Analysis. Human skeletal muscle was obtained with the absence of a stop codon, the short 5Ј untranslated region from biopsies of patients without neuromuscular disorders.

Mizuno et al. PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 ͉ 6157 Downloaded by guest on September 26, 2021 mounted on Formvar-coated carbon grids, and washed three times in PBS containing 0.5% BSA and 0.15% glycine (pH 7.4) (buffer A). This procedure was followed by a 30-min incubation with purified goat IgG (50 mg͞ml) at 25°C and three additional washes with buffer A. All of the preceding steps were designed to ensure minimal nonspecific reactions to the antibodies used. Sections then were incubated for 60 min with primary antibody (either a rabbit polyclonal antibody directed to DMN-2 or to desmin), followed by three washes in buffer A and a 60-min incubation in gold-labeled second antibody (1–2 mg͞ml). The sections were then washed six times (5 min per wash) in buffer A and then rewashed thoroughly in buffer A (five changes) and in PBS (three changes), followed by a brief fixation step in 2.5% glutaraldehyde in PBS to ensure that the antibodies did not dissociate. Subsequent steps included three further washes in PBS, five washes in water, and counterstaining with uranyl acetate and embedding in 1.25% methylcellulose. Observation was done with a Jeol 1210 electron microscope. Results Identification of an ␣-Dystrobrevin-Interacting Protein. To identify proteins that interact with ␣-dystrobrevin, we screened a human skeletal muscle cDNA library with the yeast two-hybrid ap- proach. The 5Ј region of ␣-dystrobrevin (nucleotides 7–1617) was subcloned into the pGBT9 bait vector as described in Materials and Methods. Of the 21 positive clones, three repre- sented either the same (3C and 4C) or overlapping (4C and 5D) sequences (Fig. 1A) from a yet uncharacterized protein. Data- base searching with clone 5D revealed that the protein contains sequence identity to 1,466 bp of KIAA0353 (22). The 5D clone also contained an additional 683 bp upstream from the KIAA0353 sequence. KIAA0353 is a partial cDNA sequence, Fig. 2. Summary of DMN, probes, and antibodies (A) and primary expression isolated from a brain cDNA library, which contains the inter- of DMN in heart and skeletal muscle (B and C). (A) DMN has a central ␣-helical mediate filament (IF) signature (VATYRALLE). Clone 4C is coiled-coil rod domain (1A to 2B), which is highly conserved; a nonhelical identical to the 5D sequence from 10 to 570. The exact rela- N-terminal head domain (10 aa); and a C-terminal tail domain (931 aa). The rod tionship between 4C, 5D, and KIAA0353 is given schematically domain contains nonhelical spacer elements. DMN structure and KIAA0353 A sequence are schematically aligned. KIAA0353 starts 573 bp downstream of in Fig. 1 . DMN but includes a region from 2882 to 3817 that was not present on any ␣ muscle cDNA clones. Locations of epitopes for anti-DMN antibodies (DMN-1 CoIP of the Novel Protein and -Dystrobrevin. To verify the results and DMN-2 indicate those used in Western blot and immunohistochemical of the two-hybrid screen, in vitro CoIP experiments were per- analyses, respectively) and probes (152 bp and 219 bp indicate those used for formed with different tagged portions of the protein (FLAG hybridization to Northern blots) are shown. (B) Northern blot analysis with the 5D-1, FLAG 5D-2, FLAG 4C) (Fig. 1B) and untagged dystro- 219-bp hybridization probe (A) detects a single 7.5-kb relatively abundant brevin exons 1–16. As a control, these proteins were also transcript in heart and skeletal muscle after a 6-h exposure. A longer exposure individually precipitated. Proteins were expressed and 35S- of the same Northern blot shows a faint doublet in brain (72 h). (C) Western labeled with the TNT quick coupled transcription͞translation blot analysis with an anti-DMN-1 antibody (A) detects an Ϸ160-kDa protein in heart and skeletal muscle but not in brain. system (Promega). After incubation with protein G-Sepharose, each 35S-labeled translated protein was immunoprecipitated with the anti-FLAG antibody and then analyzed by SDS͞PAGE Muscle sections were fixed in cold methanol for 3 min, blocked gel (Fig. 1B). The anti-FLAG antibody is able to specifically in 10% FCS in 1ϫ PBSfor1hat4°C, and stained with immunoprecipitate each protein individually (Fig. 1B, lanes anti-DMN-2 antibody (Fig. 2A) overnight at 4°C. The slides were 1–4). When dystrobrevin was simultaneously expressed with washed in 1ϫ PBS for 1 h and incubated with a Cy3-conjugated either FLAG 5D-2 (lane 6) or FLAG 4C (lane 7), the anti-FLAG Affinipure donkey anti-rabbit IgG (H ϩ L) secondary antibody antibody did not coimmunoprecipitate dystrobrevin to a greater degree than that seen in lane 4, where there were no FLAG (Jackson ImmunoResearch) for1hatroom temperature. The epitopes. However, when dystrobrevin and FLAG 5D-1 were slides were washed in 1ϫ PBS for 1 h and mounted with a simultaneously expressed (lane 5), dystrobrevin was coimmuno- ProLong antifade kit (Molecular Probes). Analysis was done precipitated, as indicated by the increase in the dystrobrevin with a Zeiss Axioplan 2 microscope. band over background (compare lanes 4 and 5). These results support an interaction between ␣-dystrobrevin and the clones Immunoelectron Microscopic Analysis. Human skeletal muscle was isolated from the yeast two-hybrid screen. obtained from biopsies of patients without neuromuscular dis- ͞ orders. After fixation in 2% paraformaldehyde 0.01% glutaral- Characterizing DMN. With the use of a 152-bp probe (Fig. 2A), a dehyde, the biopsies were cut into small (1-mm cubes) before phage human skeletal muscle cDNA library was screened by immersion in 2.3 M sucrose͞0.1 M PBS. The samples subse- hybridization to obtain the full-length DMN cDNA. DMN’s quently were mounted on cutting stubs, shock-frozen, and stored approximate transcription start site was identified by comparing in liquid nitrogen. Thin sections (70–100 nm) were cut with a the 5Ј sequence of the 16 DMN phage clones with that of the Reichert Ultracut S Ultramicrotome with a FC4S cryo- clones isolated from the yeast two-hybrid screening (clones 4C attachment. Sections were lifted in a small drop of sucrose, and 5D) and that obtained by two experiments with 5Ј rapid

6158 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153298 Mizuno et al. Downloaded by guest on September 26, 2021 of clone 5D-1 and recloned it back into pFHR2. Coincidentally, 5D-1 encodes the whole rod domain for DMN such that its expression from pFHR2 would encode the protein FLAG 1A- 1B-2A-2B (amino acids Ϫ1 to 337). To refine the interacting domain between DMN and ␣-dystrobrevin, we performed CoIP experiments with subfrag- ments of the two proteins. The following fragments of ␣-dys- trobrevin were amplified by PCR and subcloned into the non- FLAG pMGT1 expression vector: (i) exons 1–14, (ii) exons 8–16 (long), (iii) exons 8–16 (short), and (iv) exons 10–16. The PCR primers used for subcloning were internal to the first and last Fig. 3. Portions of the DMN rod domain specifically interact with dystrobre- listed ; consequently, these exons are not complete. The vin. (A) CoIP experiments using the anti-FLAG antibody show that FLAG exons 8–16 (long) are 23 aa longer at the N terminus than the 1A-1B-2A-2B DMN specifically interacts with dystrobrevin exons 1–16 (lane 2) and 8–16 (long) (lane 3). Other regions of ␣-dystrobrevin do not coimmuno- exons 8–16 (short). In each lane, the proteins were labeled with 35 precipitate in the presence of DMN (lanes 1, 4, and 5). (B) By varying the length [ S]methionine and expressed simultaneously with the TNT of the DMN rod domain, CoIP experiments show that dystrobrevin exons 1–16 quick coupled transcription͞translation system. CoIP experi- interacts with 1A-1B-2A-2B (lane 1) and 1A-1B-2A (lane 2), but not with the ments were performed by coexpressing FLAG 1A-1B-2A-2B shorter versions of the DMN rod domain (lanes 3–5). Proteins are identified DMN with different dystrobrevin fragments and immunopre- by an *. cipitating with the anti-FLAG antibody (Fig. 3A). Our results show that FLAG 1A-1B-2A-2B interacts specifically with dys- trobrevin exons 1–16 (lane 2) and dystrobrevin exons 8–16 (long) amplification of cDNA ends (data not shown). Most of the clones Ј (lane 3) but not exons 1–14 (lane 1), exons 8–16 (short) (lane 4), fell within 10–20 bases 5 of one another. Comparing the DMN and exons 10–16 (lane 5). These results imply that dystrobrevin cDNA sequence with that of genomic DNA (Homo sapiens, exons 8–16 are important for interactions with DMN. clone RP11–28O17), a stop codon was found 39 bases upstream We also wanted to determine which portions of the DMN rod of the predicted transcription start site and 150 bases upstream domain interact with dystrobrevin. To perform this experiment, of the first ATG codon, suggesting that this ATG likely serves we subcloned various portions of the DMN rod domain into as the translation start site. In addition, the sequence GGC pFHR2 so that they would be expressed with the N-terminal AAG ATG CTG contains an adequate Kozak consensus FLAG epitope. CoIP experiments were done by simultaneous sequence (23). translation of dystrobrevin exons 1–16 with truncated versions of BLAST analysis with DMN cDNA revealed that it is an IF the DMN rod domain (Fig. 3B). These experiments show that family member protein (24). DMN has a unique 10-aa N- binding requires the DMN domain 1A-1B-2A, although the terminal domain, followed by the hallmark rod domain, which is broken into regions 1A, 1B, 2A, and 2B and a long 931-aa inclusion of 2B adds some stability to the interaction (compare C-terminal domain. The ORF (3,762 bp) encodes a 1,253-aa lanes 1 and 2). When DMN’s entire 2A-2B, 1A-1B, or flanking protein. Compared with the KIAA0353 sequence, which was 1A and 2B regions were deleted, all interactions were either isolated from neuronal cells, muscle DMN cDNA contains an abolished or greatly diminished (lanes 3–5). additional sequence at the 5Ј end and lacks KIAA0353 region 2882–3817 (Fig. 2A). DMN Is Present at the Z-lines. To get a better idea of DMN’s subcellular localization within normal muscle, the anti-DMN-2 DMN Is Expressed Predominantly in Heart and Skeletal Muscle. To antibody was used in immunofluorescent studies. As shown in determine DMN’s expression profile, multitissue Northern blot Fig. 4E, DMN was detected as a mosaic in cross sections with analysis was performed with a 219-bp hybridization probe (Fig. minimal sarcolemmal labeling. The expression of DMN was 2A). A 7.5-kb transcript was observed in heart and skeletal stronger for type 2 fast fibers (data not shown). In longitudinal muscle after a 6-h exposure (Fig. 2B). Interestingly, a faint sections, the fibers stain in a stripe-like pattern (Fig. 4F). These doublet also was detected in brain after long exposures (72 h) results are consistent with DMN’s being an IF protein. (Fig. 2B). With the use of the 152-bp DMN probe, the same Immunoelectron microscopy was used to validate the putative probe that was used for screening the phage cDNA library, DMN Z-line staining seen by immunofluorescence (Fig. 4). Fig. 4 A–C mRNA was again detected in both heart and skeletal muscle. No shows typical staining for DMN in skeletal muscles from three doublet was detected in brain with this probe, even after long different patients without neuromuscular diseases. DMN ap- pears to be localized between adjacent at the level of exposures (data not shown). The doublet in brain detected CELL BIOLOGY with the 219-bp probe suggests that there may be tissue-specific the Z-lines. To confirm this, an equivalent experiment was DMN isoforms. performed on the same muscle biopsies with the use of anti- Sequence analysis of DMN’s cDNA predicts that the encoded bodies to desmin (Fig. 4D), which envelops all myofibrils at the protein would have a molecular mass of Ϸ140 kDa. Western blot Z-lines. Interestingly, we find that DMN colocalizes with desmin. analysis with a DMN-1-specific antibody (Fig. 2A) detected a protein calculated to be 160 kDa, expressed solely in heart and DMN Rod Domain Interacts with Desmin. Because DMN and desmin skeletal muscle (Fig. 3C), thereby supporting the results of the colocalize, we checked for an interaction between DMN and Northern blot. desmin with CoIP experiments. These experiments were done by coexpressing DMN (FLAG 1A-1B-2A-2B, FLAG C-terminal-1, DMN Rod Domain Interacts with Exon 8–16 of ␣-Dystrobrevin. By or FLAG C-terminal-2) with untagged desmin and then immu- both yeast two-hybrid and CoIP analyses, FLAG 5D-1 (amino noprecipitating the 35S-labeled proteins with the use of the acids Ϫ37 to 337) interacts with dystrobrevin exons 1–16 (Fig. 1). anti-FLAG antibody (Fig. 5A). Our results show that desmin As a consequence of cloning 5D-1 with its 5Ј untranslated region coimmunoprecipitates with FLAG 1A-1B-2A-2B (lane 1) but downstream of the N-terminal FLAG, this construct contains an not with either FLAG C-terminal-1 (lane 2) or FLAG C- additional 37 aa before the initiation methionine. To eliminate terminal-2 (lane 3). To further define which portion of the DMN the possibility that this small segment affected our rod domain interacts with desmin, we simultaneously expressed results, we removed the 5Ј untranslated region from the cDNA desmin and truncated versions of the FLAG DMN rod domain

Mizuno et al. PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 ͉ 6159 Downloaded by guest on September 26, 2021 Table 1. Comparison of DMN with other major IF and IF-associated proteins No. of residues

N C Homology Type Protein terminus Rod terminus with DMN, %

1 Human 14 114 312 46 41 2 Human 167 314 109 43 3 Human desmin 107 309 54 49 4 Human neurofilament M 100 312 504 47 5 Human C 30 357 185 40 6 Rat 7 307 1,491 49 Frog tanabin 12 308 1,424 50 Chicken paranemin 15 308 1,283 51 Chicken 10 304 1,290 60 Human DMN 10 312 931 100

Representative proteins from each of the different IF classes are listed for comparison with human DMN. The sum of identical and similar amino acids is given for only the conserved rod domain of DMN and the other IF proteins. IF-associated but unclassified proteins are blank for IF protein type.

(27), and chicken synemin (28), all of which have a short N-terminal and long C-terminal domains. These proteins, to- gether with nestin, should be classified as a type 6 IF protein or even a type 7 IF protein. Comparing DMN’s rod domain sequence with other IF proteins, human DMN has 60% homol- ogy with chicken synemin, indicating that DMN is more similar Fig. 4. DMN is located in the Z-lines. When muscle was stained with anti- to chicken synemin than it is to rat nestin, frog tanabin, and DMN-2 antibody, a mosaic staining pattern was detected in cross sections (E), chicken paranemin. DMN is not likely to be the human ortholog and a stripe-like staining pattern was detected in longitudinal sections (F). of chicken synemin because other IF proteins cloned in both Three different muscle tissues were stained for DMN (A–C), thereby localizing species, for example , are greater than 80% homologous DMN to the filamentous structures running between the Z-lines (Z) (arrow- to one another. heads). These tissues are the regions that also stain for desmin (D) (arrow- heads). (Bar ϭ 200 nm.) Discussion ␣-Dystrobrevin 1 is the largest of the five ␣-dystrobrevin splice variants. We used the first 16 of the 21 ␣-dystrobrevin exons as (Fig. 5B). This CoIP experiment shows that desmin interacts with the bait in a yeast two-hybrid screen to clone desmuslin. Because all of the rod domain constructs we generated (lanes 1–5), these exons are also shared with ␣-dystrobrevin 2, it is likely that suggesting that any portion of this domain is sufficient for DMN can also interact with both of these ␣-dystrobrevin interaction. isoforms. Sequence analysis suggests that DMN contains an IF signature Comparison of DMN with Other IF Proteins. DMN and various other (24), suggesting that DMN may be a structural protein. Of the IF proteins are grouped in Table 1, along with three IF- various IF protein family members, DMN is most similar to associated proteins. Amino acid prediction analysis was per- synemin (28), paranemin (27), tanabin (26), and nestin (25) in formed with MACVECTOR software (Oxford Molecular Group). terms of domain structure. All have short N-terminal, conserved When the amino acid numbers of N-terminal, rod, and C- rod, and long C-terminal domains (Table 1). Currently, nestin is terminal domains were compared, human DMN most closely the only one classified as a type 6 IF protein. The others are resembled rat nestin (25), frog tanabin (26), chicken paranemin either not yet classified or are called IF-associated proteins, although Steinert et al. (29) have proposed that they all be grouped as type 6 IF proteins. By domain structure, we believe that DMN should also be grouped as a type 6 IF protein. Like DMN, chicken synemin also shares homology with parts of KIAA0353. Although several parts of synemin’s C-terminal domain and the extra C-terminal end (50 aa) are almost identical to KIAA0353 (28), the remainder is not homologous. On the other hand, DMN shares 100% homology with the entire KIAA0353 cDNA, except at the 5Ј terminus, where DMN has an additional 572 bases, and at the 3Ј terminus, where DMN lacks region 2882–3817. However, unlike chicken synemin, our in vitro CoIP experiments show that the C-terminal domain of DMN does not interact with ␣- (data not shown). Because of Fig. 5. Interaction of DMN with desmin. (A) CoIP analysis shows that the DMN FLAG 1A-1B-2A-2B subfragment (lane 1) interacts with desmin, whereas differences in binding preference and sequence homology, we do the DMN FLAG C-terminal subfragments do not (lanes 2 and 3). (B) CoIP not believe that DMN is the human homolog of chicken synemin. experiments with full-length human desmin show that desmin interacts with Recently, , an IF protein that interacts with all five truncated FLAG DMN constructs (lanes 1–5). Desmin (identified with ␣-dystrobrevin, was reported to be concentrated at the neuro- an *) was 35S-labeled. muscular junction of normal skeletal muscle (30). In dystrophic

6160 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153298 Mizuno et al. Downloaded by guest on September 26, 2021 muscle, syncoilin’s expression was increased such that the pro- reinforcing protein connections. Desmin encircles Z-lines and tein was found throughout the . This finding is in then makes longitudinal filamentous bridges between neighbor- contrast to DMN, which is expressed similarly in control and ing Z-lines. These bridges are supported by plectin (31) and DMD muscle (data not shown). This difference in their expres- ␣B-crystallin (32). Plectin links desmin to the Z-discs and desmin sion profile suggests that DMN and syncoilin have different to itself (31). in the desmin gene can cause desmin functions. Moreover, syncoilin shares the greatest homology characterized by muscle weakness, cardiac impair- with type 3 and 4 IF proteins rather than a type 6 IF protein like ment, and intracytoplasmic accumulation of desmin deposits DMN. These results suggest that DMN is a yet uncharacterized (33–36). The accumulation of desmin may actually be a second- protein present in muscle. ary feature, as other proteins also accumulate in deposits. Two observations led us to identify proteins other than Although we have not yet investigated this possibility, it is ␣-dystrobrevin that are capable of interacting with DMN. First, possible that DMN deposits would be found in muscle from a electron microscopic analysis colocalized DMN with desmin, desmin , and͞or desmin deposits may be found in another IF group family member. Second, desmin had been shown to be capable of interacting with synemin (28), a protein muscle with mutations in the DMN gene. In summary, we have cloned and subsequently characterized that shares some homology with DMN. As such, we attempted ␣ to determine whether DMN could interact with desmin with the an -dystrobrevin-interacting protein, which we have termed use of CoIP experiments. As expected, DMN can interact with desmuslin (DMN). Our results support a model in which DMN desmin, and the site of interaction was localized to DMN’s rod forms a linkage connecting the extracellular matrix to the domain (Fig. 5A). ␣-Dystrobrevin, a , also Z-discs. The DMN linkage we have identified is a linkage interacts with DMN at the rod domain. Therefore, we speculate between a component of dystrophin-associated protein complex that there are at least two distinct DMN subpopulations, one in (␣-dystrobrevin) and a component of the Z-lines (desmin). This which DMN interacts exclusively with desmin within the Z-lines, linkage can be extended to the extracellular matrix by recogniz- and another in which DMN interacts with both dystrobrevin and ing that the extracellular matrix protein laminin interacts with desmin at the . It is possible that posttranslational ␣-dystroglycan (2). Disruption of any of the proteins in this modifications or other interacting proteins could modulate linkage could potentially damage muscle cell integrity. DMN’s intracellular location. IF proteins like DMN and desmin are thought to help S.C.W. is supported by a grant from the Muscular Dystrophy Associa- maintain the structural integrity of tissues by mechanically tion. L.M.K. is an investigator of the Howard Hughes Medical Institute.

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