Dynamic Changes in the Subcellular Localization of Drosophila Β-Sarcoglycan During the Cell Cycle

Dynamic Changes in the Subcellular Localization of Drosophila β-Sarcoglycan during the Cell Cycle

Reina Hashimoto1 and Masamitsu Yamaguchi1∗ 1Department of Applied Biology and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

ABSTRACT. One of the proposed roles of sarcoglycan is to stabilize dystrophin glycoprotein complexes in muscle sarcolemma. Involvement in signal transduction has also been proposed and abnormalities in some sarcoglycan genes are known to be responsible for muscular dystrophy. While characterization of sarcoglycans in muscle has been performed, little is known about its functions in the non-muscle tissues in which mammalian sarcoglycans are expressed. Here, we investigated temporal and spatial expression patterns of Drosophila β-sarcoglycan (dScgβ) during development by immunohistochemistry. In addition to almost ubiquitous expression in various tissues and organs, as seen for its mammalian counterpart, anti-dScgβ staining data of embryos, eye imaginal discs, and salivary glands demonstrated cytoplasmic localization during S phase in addition to plasma membrane staining. Furthermore we found that subcellular localization of dScgβ dramatically changes during mitosis through possible association with tubulin. These observations point to a complex role of sarcoglycans in non-muscle tissues.

Key words: sarcoglycan/Drosophila/tubulin/cell cycle

Introduction plex is to stabilize DGC itself by interacting with dystrophin, dystroglycan, and other constitutive proteins (Chen et β-sarcoglycan is a member of the sarcoglycan complex. al., 2006; Crosbie et al., 1999; Durbeej et al., 2000; Straub This forms part of the dystrophin glycoprotein complex et al., 1998). The β-/δ-sarcoglycan core is known to interact (DGC) components, which have been seen to be mutated in with the C-terminus of dystrophin (Chen et al., 2006). Bio- muscular dystrophy (Ozawa et al., 2005). Sarcoglycan com- chemical preparations from sarcoglycan mutant muscle plexes are heteromeric, consisting of α-, β-, γ-, δ-, ε-, and ζ- have revealed less tightly adherent α-dystroglycan subunits sarcoglycans in humans (Ettinger et al., 1997; Lim et al., suggesting abnormal interaction in the absence of sarco- 1995; Nigro et al., 1996; Noguchi et al., 1995; Roberds et glycan (Durbeej et al., 2000; Straub et al., 1998). Further- al., 1994; Wheeler et al., 2002). Abnormalities in the α-, β-, more, lack of sarcoglycans has been demonstrated to cause γ-, and δ-sarcoglycans genes are responsible for autosomal instability of the DGC in several sarcoglycan-null mouse recessive limb girdle muscular dystrophies (LGMD2D-2F) models (Durbeej and Campbell, 2002). Moreover, recent (Ozawa et al., 2005; Vainzof et al., 1996), and muta- studies have indicated that sarcoglycans might also have tions in the ε-sarcoglycan encoding gene are associated non-mechanical functions, for example, functionally com- with myoclonus-dystonia syndrome of neurological origin pensating for integrins in muscle (Allikian et al., 2004). (Zimprich et al., 2001). α-Sarcoglycan has an ATP-binding site in the extracellular One of the proposed functions of the sarcoglycan com- region, and α-sarcoglycan-transfected HEK293 cells exhibit a significant increase in ATP-hydrolyzing activity that is abolished by anti-α-sarcoglycan antibodies, suggesting *To whom correspondence should be addressed: Masamitsu Yamaguchi, the existence of ecto-ATPase activity (Betto et al., 1999; Department of Applied Biology, Kyoto Institute of Technology, Matsu- gasaki, Sakyo-ku, Kyoto 606-8585, Japan. Sandona et al., 2004). It has also been reported that sarco- Tel: +81–75–724–7781, Fax: +81–75–724–7760 glycans associate with integrins and nNOS, indicating a E-mail: [email protected] potential role in signaling transduction. In fact, we have Abbreviations: dScgβ, Drosophila β-sarcoglycan; DGC, dystrophin glyco- β protein complex; LGMD, limb girdle muscular dystrophy; PI, propidium shown that -sarcoglycan negatively regulates the Egfr sig- iodide. naling pathway (Hashimoto and Yamaguchi, 2006). These

173 R. Hashimoto and M. Yamaguchi reports suggest that sarcoglycans are multifunctional pro- previously (Hashimoto and Yamaguchi, 2006), and used at 1:4,000 teins. dilution for Western immunoblot analysis, and at 1:200 dilution To date, the function of DGC in muscle has been exten- for immunohistochemistry. Mouse monoclonal anti-α-tubulin and sively studied. However, despite current evidence that com- anti-lamin antibodies (Developmental Studies Hybridoma Bank) ponents of DGC are ubiquitously expressed in various were both used at 1:400 dilution for immunohistochemistry, and mammalian tissues such as retina, brain, blood-brain bar- anti-PCNA antibodies (Sigma) were used at 1:2,000 dilution for rier, and kidney (Chan et al., 2005; Culligan et al., 1998; Western immunoblotting. Dalloz et al., 2001; Fort et al., 2005; Lien et al., 2006; Sekiguchi, 2005), roles in such non-muscle tissues remain Western immunoblot analysis obscure. Their elucidation should help in understanding DGC and the pathogenesis of muscular dystrophies. Third instar larval extracts from Canton S and transgenic flies To assess DGC functions, various animal models have carrying Act5C-GAL4>UAS-dscgβ and Act5C-GAL4>UAS- been utilized. In addition to rodents such as mice, rats and IR-dscgβ or embryonic extracts from Canton S were applied to hamster, C. elegans and zebrafish are currently used for SDS-polyacrylamide gels containing 7.5% or 10% acrylamide and research into human diseases (Bassett and Currie, 2003; proteins were transferred to polyvinylidene difluoride membranes Chamberlain and Benian, 2000). For instance, in the δ- (Bio-Rad). The blotted membranes were blocked with TBS-T con- sarcoglycan anti-sense morpholin-injected zebrafish, other taining 2% bovine serum albumin, followed by incubation with the associated sarcoglycans are downregulated, while dystro- primary antibodies for 16 h at 4°C. After washing, the membranes phin expression is not affected. A similar phenotype is typi- were incubated with HRP conjugated secondary antibodies (Amer- cal of human patients carrying mutations in δ-sarcoglycan, sham Biosciences) at 1:5,000 dilution for 1 h at 25°C. Detection suggesting that the zebrafish is an excellent model for the was performed with ECL Western blotting detection reagent and analysis of the limb-girdle muscular dystrophy (Guyon et images were analyzed with a Lumivision Pro HSII image analyzer al., 2005). Orthologs of some of the DGC members are also (Aisin Seiki). found in C. elegans (Grisoni et al., 2002) Recently, Drosophila has also been utilized as an animal Immunohistochemistry model to study human diseases (Celotto and Palladino, 2005; O’Kane, 2003). The majority of the mammalian DGC Canton S embryos were collected, dechorionated in a 50% Clorox protein subclasses is well conserved in Drosophila (Greener solution, washed with 0.7 M NaCl/0.2% Triton X-100 and fixed in and Roberts, 2000), but little is known whether their pro- a 1:1 heptane: 4% paraformaldehyde in TBS mixture for 20 min perties and functions are also evolutionary conserved. To with vigorous shaking at 25°C. Vitelline membranes were then address this question, we here investigated the expression removed by shaking embryos in heptane-methanol at 25°C for 30 pattern of Drosophila β-sarcoglycan (dScgβ) protein in var- sec. Embryos were blocked with TBS containing 10% goat serum ious tissues and its subcellular localization. In the present and 0.15% Triton X-100 (TBT) before incubation with the primary report we document further evidence for the validity of antibodies in the same solution for 16 h at 4°C. The embryos were Drosophila as a model animal in which to study limb-girdle washed extensively in TBS containing 0.3% Triton X-100, muscular dystrophy, showing that the expression pattern of reblocked with TBT and incubated with the secondary antibody for Drosophila β-sarcoglycan in various tissues is similar to 3 h at 25°C. After extensive washing with TBS containing 0.3% that of mammalian β-sarcoglycan. Furthermore, dScgβ was Triton X-100, they were mounted and observed using a Zeiss found to localize in cytoplasm in addition to plasma mem- LSM510 confocal laser scanning microscope. For propidium branes, with dramatic changes depending on the cell cycle. iodide (PI) staining, embryos after fixation were treated with 200 µg/ml RNAase for 3 h at 37°C, washed extensively, and stained with 20 µg/ml PI for 2 h at 25°C. For phalloidin staining, embryos Materials and Methods after fixation were incubated with 1 unit/200 µl phalloidin for 20 min at 25°C. Fly stocks Third instar larvae of Canton S were dissected and the tissues were fixed with 4% paraformaldehyde in TBS mixture for 20 min Fly stocks were maintained at 25°C on standard food containing at 25°C. The steps after fixation were the same as for the protocol 0.7% agar, 5% glucose and 7% dry yeast. Canton S was used as the for immunohistochemistry of embryos. wild type. Transgenic flies carrying Act5C-GAL4>UAS-dscgβ and Act5C-GAL4>UAS-IR-dscgβ were established as described previously (Hashimoto and Yamaguchi, 2006). Results and Discussion

Antibodies Specificity of the anti-dScgβ antibody Rabbit polyclonal anti-dScgβ antibodies were produced as described With antibodies against the full length dScgβ, two major

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bands could be detected on Western blots of Flag-dScgβ- over-expressed larval extracts (Fig. 1A, lane 1 and 2), with estimated molecular weights of 48 kDa and 114 kDa. These two bands could also be observed with anti-Flag antibodies, indicating that they are exogenously over-expressed dScgβ (Fig. 1A, lane 1 and 2). Based on amino acid sequence, dScgβ would be expected to migrate at 38,114. The slower migration observed here on SDS-PAGE is likely to be due to its unusual amino acid composition. The 114 kDa band may represent a dimeric or trimeric form of dScgβ. Dimer or multimer formation in the presence of SDS has been reported for mammalian sarcoglycans (Shi et al., 2004). It has also been reported that some cell surface antigens such as HLA-DR, -DC1 and -B7 form dimeric or multimeric forms even in the SDS-PAGE (Signas et al., 1982; Bono and Strominger, 1982). Anti-dScgβ antibodies could also detect a weak band at around 54 kDa in extracts from both Flag-dScgβ over-expressing and wild type larvae (Fig. 1A, lane 2 and 3). Moreover, this band was largely reduced in the larval extract from the dScgβ knock down flies (Fig. 1B). The data thus suggest that although this weak 54 kDa band is larger than expected, it does represent endogenous dScgβ. The discrepancy is likely to be due to some post- translational modification of dScgβ, such as phosphorylation and glycosylation, as reported for mammalian β- sarcoglycan (Shi et al., 2004).

Expression pattern of dScgβ during embryogenesis Developmental Western blot analysis during embryogenesis showed ubiquitous expression of dScgβ (Fig. 1C). Levels of dScgβ protein were highest in unfertilized embryo extracts, suggesting abundant maternal storage of dScgβ (Fig. 1C, lane 1). These data are consistent with strong anti-dScgβ immunostaining in ooplasm (Fig. 3C). Immunohistochemis- try of the whole mount embryos showed dScgβ protein to be highly expressed in midgut (Fig. 2A) and the muscula- Fig. 1. Western immunoblot analysis. (A, B) Specificity of the affinity ture, including dorsal and ventral oblique muscles (Fig. 2B), β purified anti-dScg antibody. Proteins in extracts of third instar larvae of pleural muscles (Fig. 2B), and the dorsal pharyngeal muscu- Act5C-GAL4>UAS-flag-dScgβ (lane 1 and 2) and Canton S (lane 3) were β separated by SDS-polyacrylamide gel electrophoresis. Lane 1 was blotted lature (Fig. 2A). In mammals, -sarcoglycan is primarily with anti-Flag antibody and lanes 2 and 3 with anti-dScgβ antibody. White expressed in skeletal muscle, smooth and cardiac muscle, in arrowheads indicate the exogenously overexpressed dScgβ and the black line with the fact that deficiency of β-sarcoglycan consti- arrowhead the endogenous dScgβ. (B) Proteins in the extracts of third tutes the primary cause of LGMD 2E (Bonnemann et al., instar larvae of Act5C-GAL4>UAS-IR-dScgβ (lane 1) and Canton S (lane 1996). The high expression of dScgβ protein in musculature 2) were separated by SDS-polyacrylamide gel electrophoresis and blotted is consistent with that of mammalian β-sarcoglycan. β with anti-dScg antibodies. The black arrowhead indicates the endogenous To date, embryonic expression patterns of Drosophila dScgβ. Reduction of dScgβ protein in RNAi flies (lane 1) is evident, indicating that the 54 kDa band represents endogenous dScgβ. (C) Devel- DGC component orthologs have been determined using opmental Western blot during embryogenesis. Proteins in the extracts RNA in situ hybridization (Dekkers et al., 2004). In the of unfertilized (lane 1), 0–4 h (lane 2), 4–8 h (lane 3), 8–12 h (lane 4), RNA level, dscgβ is expressed in brain, ventral nerve cord, 12–16 h (lane 5), and 16–20 h (lane 6) embryos were separated by SDS- and midgut (Dekkers et al., 2004). However, dScgβ protein polyacrylamide gel electrophoresis and blotted with anti-dScgβ antibodies. was not detected in brain and ventral nerve cord (Fig. 2A). Unfertilized embryos (lane 1) show maternal storage. In all experiments These differences can be explained by variation in the turn- for this figure, PCNA was used as a total protein loading control. over rate of dScgβ protein in different tissues. In stage 17 embryos, the musculature is already differentiated, while the neural tissues such as brain and ventral nerve cord are

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sarcoglycan proteins is accompanied by progress of C2C12 cell differentiation (Noguchi et al., 1999). Therefore, it is likely that the turnover rate of dScgβ is very rapid in proliferating cells like embryonic neural tissues but slow in differentiated cells such as the musculature in Drosophila. Furthermore, we found that dScgβ is also expressed in epithelial cells of embryos (Fig. 2C), larval imaginal discs (Fig. 2D–F), and salivary glands (Fig. 2G). In mammals, sarcoglycan complexes are present not only in muscle but also in various non-muscle tissues such as the retina and endothelium (Claudepierre et al., 2000; Dalloz et al., 2001; Fort et al., 2005; Ramirez-Sanchez et al., 2005). Also in zebrafish, in situ hybridization analysis has shown that δ- sarcoglycan mRNA can be detected in brain and retina, in addition to the primary detection in the skeletal and cardiac muscles (Cheng et al., 2006). The varied expression of sarcoglycans is therefore conserved among species, suggesting multifunctional roles for sarcoglycans. We therefore investigated the intercellular localization of dScgβ in eye imaginal discs by double staining with anti- dScgβ antibody and phalloidin to visualize plasma membrane by binding filamentous actin, or anti-lamin antibodies to visualize nuclear membranes. We found dScgβ to be localized both in plasma membranes (Fig. 2H) and in cytoplasm (Fig. 2I). Previous immunohistochemistry data for myotube cultures from normal human muscle showed an intercellular localization for β-sarcoglycan at early stages of myoblast fusion. It subsequently shifts to many spots in the perinuclear area, and finally localizes at the sarcolemma (Radojevic et al., 2000). Thus dScgβ may have a role not only on plasma membranes but also in the cytoplasm.

Sub-cellular distribution of dScgβ during the cell cycle The predominant localization of mammalian DGC compo- Fig. 2. Expression pattern of dScgβ during development. (A–C) whole nents in plasma membranes is well known. On the other mounts of staged embryos were stained with anti-dScgβ antibody. Arrow- hand, several recent studies have revealed that mammalian heads and arrows in panel A indicate the midgut and dorsal pharyngeal DGC components are also present in cytoplasm and nuclei musculature, respectively. Arrowheads and arrows in panel B show pleural (Calderilla-Barbosa et al., 2006; Fuentes-Mera et al., 2006; muscles and oblique muscles, respectively. dScgβ is also expressed in epithelial cells of embryos as shown in panel C. (A and B) dorsal view. (C) Marquez et al., 2003; Radojevic et al., 2000). However, no lateral view. (D–G) Imaginal discs and salivary gland are stained with anti- study which addresses their dynamics of distribution on cell dScgβ antibodies. In all tissues tested here, anti-dScgβ signals were cycle progression has been reported so far. detected. (H, I) Enlarged images of eye imaginal discs. Anti-dScgβ signals Drosophila provides examples of many distinct cell cycle are shown in left panels of H and I. The merged images of anti-dScgβ and patterns all in a single organism. During embryogenesis, the phalloidin or anti-lamin signals are shown in right panels of H and I, first 13 cycles show nearly synchronous nuclear division β respectively. dScg is localized in cytoplasm (panel I) in addition at consisting of S and M phases. After cellularization, most plasma membrane (panel H). Bars in panel A–G indicate 100 µm, and bars in panels H and I indicate 20 µm. epidermal cells undergo three more cell cycles (cycles 14– 16) that are regulated by S/G2/M transition. Following mitosis 16, most embryonic cells enter an extended G1 phase. still proliferating. In mammalian systems, it is reported that Some of the G1-arrested cells such as imaginal discs that in proliferating rat C2C12 cultured myocytes, mRNAs for give rise to the adult body resume G1/S/G2/M cell cycles α-, β-, γ-, and δ-sarcoglycans are readily detectable while only after the larva hatches. Other G1-arrested cells sub- levels of proteins are very low, indicating a high turnover sequently resume rounds of endoreplication (S/G cycle), rate in the proliferating cells (Noguchi et al., 1999). Further- giving rise to polyploid or polytene larval cells such as more, it has also been demonstrated that accumulation of cells in the salivary glands.

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Fig. 3. Expression pattern of dScgβ during early embryonic development. (A) Normalski image of (B). (B) Negative staining control without first antibody. No anti-dScgβ signals being observed. (C) Whole mount of early stage embryos stained with anti-dScgβ antibody. Early embryos before cellularization (S/M cycle) appear to have abundant maternal storage of dScgβ protein. (D) Whole mount of stage 6 embryo stained with anti-dScgβ antibody. Broken-line circles show examples of mitotic domains after cellularization (S/G2/M cycle) where anti-dScgβ signals at plasma membranes are diminished. All bars indicate 100 µm.

Firstly we examined the dScgβ distribution in embryos detect any localization change of dScgβ during M phase. before cellularization. dScgβ was detected in the whole We next examined the behavior of dScgβ in eye imaginal embryo, suggesting maternal storage in the ooplasm (Fig. discs of third instar larvae (Fig. 4C), where typical G1/S/G2/ 3C). In cycle 14, cellularization starts with S/G2/M type M type cell cycles prevail. As observed in embryos, dScgβ regulation, as described above, and group of cells, termed was localized in cytoplasm in addition to plasma mem- mitotic domains, enter mitosis in close synchrony with each branes during S phase (Fig. 4C). Again, it was also seen to other, but out of synchrony with cells of other mitotic be partially co-localized with tubulin during M phase (Fig. domains. The anti-dScgβ staining data revealed a non- 4C), suggesting that the dynamic change of dScgβ localiza- plasma membrane localization of dScgβ in mitotic domains tion during mitosis appears to be common among different (Fig. 3D). PI staining allowed us to distinguish each phase tissues. It is well known that DGC closely interacts with the of the cell cycle easily. Intriguingly, we found that the actin cytoskeleton through dystrophin. However, an interac- subcellular distribution of dScgβ is dramatically changed tion with tubulin has not been previously reported. The dependent on the cell cycle in embryos (Fig. 4A). In S phase present work is the first report suggesting their possible cells, dScgβ is localized in the cytoplasm and plasma mem- interaction. branes (Fig. 4A). In M phase cells, it moves away from the Finally we examined the salivary glands where cells are membranes, appearing to associate with the ends of the regulated by S/G type cell cycling. dScgβ was predomi- chromosomes, as if regulating chromosomes segregation nantly localized at plasma membranes, as observed with and cytokinesis (Fig. 4A). The anti-dScgβ staining pattern other tissues (Fig. 4D), with a reticulated pattern in the in M phase cells is very similar to that of tubulin. Therefore, cytoplasm and perinuclear localization also being clearly we carried out double staining of embryos with anti-dScgβ observed. In addition, dScgβ localization in the nuclear and anti-α-tubulin antibodies. The data showed at least par- matrix was also detected (Fig. 4D). tial co-localization of the two (Fig. 4B). In embryos before In S phase cells of all tissues tested here, we observed cellularization, the high background makes it difficult to cytoplasmic distribution of dScgβ in addition to localization

177 R. Hashimoto and M. Yamaguchi

Fig. 4. Sub-cellular distribution of dScgβ during the cell cycle. (A and B) Enlarged images of cells in mitotic domains (S/G2/M cycle) are shown. Red and green indicate PI and anti-dScgβ signals, respectively in panel A, while in panel B, red and green indicate anti-tubulin and anti-dScgβ signals, respectively. Arrowheads in panel B show examples of partial co-localization between dScgβ and tubulin. (C) An eye imaginal disc (G1/S/G2/M cycle) double stained with anti-tubulin (red) and anti-dScgβ antibodies (green). The merged image is shown in the right panel. Arrowheads in panel E show examples of cells undergoing mitosis. Partial co-localization of dScgβ and tubulin was observed also in eye imaginal discs. (D) Salivary gland (S/G cycle) double stained with anti-dScgβ antibodies (red) and phalloidin (green). The merged image is shown in the right panel. Arrows indicate plasma membrane localization of dScgβ while arrowheads show perinuclear localization. A reticulated pattern in the cytoplasm is also evident. Bars in panel A, panel B, panel C, and panel D indicate 2,5 µm, 10 µm, 5 µm, and 20 µm, respectively. at plasma membranes (Fig. 4). Furthermore, at least partial (Calderilla-Barbosa et al., 2006). However, the roles of co-localization of dScgβ and tubulin was observed in M these proteins in nuclei remain to be elucidated. In the phase cells. These data taken together suggest that dScgβ present study, we found that dScgβ changes its localization might play a positive role in onset or progression of mitosis coupled with cell cycle progression. We also suggest that the in association with tubulin. It is interesting to examine the movement of dScgβ during mitosis may due to the associ- effect of dScgβ knock down on the progression of the cell ation with tubulin. Our findings point to further functions cycle. However, it has so far proved to be technically diffi- of some DGC components in nuclei. cult to knock down dScgβ in proliferating cells and tissues, most likely because of the relatively long half-life of the Acknowledgements. We are grateful to Drs M. Moore and S. Cotterill for dScgβ protein and a high abundance of dScgβ mRNA in comments on the English manuscript. This work was supported by grants these cells (Hashimoto and Yamaguchi, 2006). Establish- from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. ment and analysis of dScgβ mutant fly lines may help to β clarify this point. The presence of -sarcoglycan in nuclei References was previously reported in HeLa cells based on confocal microscopy and also its association with the nuclear matrix. Allikian, M.J., Hack, A.A., Mewborn, S., Mayer, U., and McNally, E.M. Similar observations have also been made for Dp71 dystro- 2004. Genetic compensation for sarcoglycan loss by integrin β α β α β alpha7beta1 in muscle. J. Cell Sci., 117: 3821–3830. phin, -dystroglycan, - and -syntrophin, 1- and - Bassett, D.I. and Currie, P.D. 2003. The zebrafish as a model for muscular dystrobrevin (Fuentes-Mera et al., 2006). Moreover, Dp71 dystrophy and congenital myopathy. Hum. Mol. Genet., 12, Spec No. 2: dystrophin is also localized in the nuclei of PC12 cells R265–270.

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Betto, R., Senter, L., Ceoldo, S., Tarricone, E., Biral, D., and Salviati, G. dent from dystrophins. Neurosci. Res., 53: 25–33. 1999. Ecto-ATPase activity of alpha-sarcoglycan (adhalin). J. Biol. Fuentes-Mera, L., Rodriguez-Munoz, R., Gonzalez-Ramirez, R., Garcia- Chem., 274: 7907–7912. Sierra, F., Gonzalez, E., Mornet, D., and Cisneros, B. 2006. Character- Bonnemann, C.G., Passos-Bueno, M.R., McNally, E.M., Vainzof, M., de ization of a novel Dp71 dystrophin-associated protein complex (DAPC) Sa Moreira, E., Marie, S.K., Pavanello, R.C., Noguchi, S., Ozawa, E., present in the nucleus of HeLa cells: Members of the nuclear DAPC Zatz, M., and Kunkel, L.M. 1996. Genomic screening for beta-sarcoglycan associate with the nuclear matrix. Exp. Cell Res., 312: 3023–3035. gene mutations: missense mutations may cause severe limb-girdle mus- Greener, M.J. and Roberts, R.G. 2000. Conservation of components of cular dystrophy type 2E (LGMD 2E). Hum. Mol. Genet., 5: 1953–1961. the dystrophin complex in Drosophila. FEBS Lett., 482: 13–18. Bono, M.R. and Strominger, J.L. 1982. Direct evidence of homology Grisoni, K., Martin, E., Gieseler, K., Mariol, M.C., and Segalat, L. 2002. between human DC-1 antigen and muraine I-A molecules. Nature, 299: Genetic evidence for a dystrophin-glycoprotein complex (DGC) in 836–838. Caenorhabditis elegans. Gene, 294: 77–86. Calderilla-Barbosa, L., Ortega, A., and Cisneros, B. 2006. Phosphorylation Guyon, J.R., Mosley, A.N., Jun, S.J., Montanaro, F., Steffen, L.S., Zhou, of dystrophin Dp71d by Ca/calmodulin-dependent protein kinase II Y., Nigro, V., Zon, L.I., and Kunkel, L.M. 2005. Delta-sarcoglycan is modulates the Dp71d nuclear localization in PC12 cells. J. Neurochem., required for early zebrafish muscle organization. Exp. Cell Res., 304: 98: 713–722. 105–115. Celotto, A.M. and Palladino, M.J. 2005. Drosophila: a “model” model Hashimoto, R. and Yamaguchi, M. 2006. Genetic link between beta- system to study neurodegeneration. Mol. Interv., 5: 292–303. sarcoglycan and the Egfr signaling pathway. Biochem. Biophys. Res. Chamberlain, J.S. and Benian, G.M. 2000. Muscular dystrophy: the worm Commun., 348: 212–221. turns to genetic disease. Curr. Biol., 10: R795–797. Lien, C.F., Hazai, D., Yeung, D., Tan, J., Fuchtbauer, E.M., Jancsik, V., Chan, P., Gonzalez-Maeso. J., Ruf, F., Bishop, D.F., Hof, P.R., and Sealfon, and Gorecki, D.C. 2006. Expression of alpha-dystrobrevin in blood- S.C. 2005. Epsilon-sarcoglycan immunoreactivity and mRNA expres- tissue barriers: sub-cellular localisation and molecular characterisation sion in mouse brain. J. Comp. Neurol., 482: 50–73. in normal and dystrophic mice. Cell Tissue Res. [Epub ahead of print] Chen, J., Shi, W., Zhang, Y., Sokol, R., Cai, H., Lun, M., Moore, B.F., Lim, L.E., Duclos, F., Broux, O., Bourg, N., Sunada, Y., Allamand, V., Farber, M.J., Stepanchick, J.S., Bonnemann, C.G., and Chan, Y.M. Meyer, J., Richard, I., Moomaw, C., Slaughter, C., and et al. 1995. 2006. Identification of functional domains in sarcoglycans essential Beta-sarcoglycan: characterization and role in limb-girdle muscular for their interaction and plasma membrane targeting. Exp. Cell Res., dystrophy linked to 4q12. Nat. Genet., 11: 257–265. 312: 1610–1625. Marquez, F.G., Cisneros, B., Garcia, F., Ceja, V., Velazquez, F., Depardon, Cheng, L., Guo, X.F., Yang, X.Y., Chong, M., Cheng, J., Li, G., Gui, Y.H., F., Cervantes, L., Rendon, A., Mornet, D., Rosas-vargas, H., Mustre, and Lu, D.R. 2006. Delta-sarcoglycan is necessary for early heart and M., and Montanez, C. 2003. Differential expression and subcellular muscle development in zebrafish. Biochem. Biophys. Res. Commun., distribution of dystrophin Dp71 isoforms during differentiation process. 344: 1290–1299. Neuroscience, 118: 957–966. Claudepierre, T., Dalloz, C., Mornet, D., Matsumura, K., Sahel, J., and Nigro, V., de Sa Moreira, E., Piluso, G., Vainzof, M., Belsito, A., Politano, Rendon, A. 2000. Characterization of the intermolecular associations of L., Puca, A.A. Passos-Bueno, M.R., and Zatz, M. 1996. Autosomal the dystrophin-associated glycoprotein complex in retinal Muller glial recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a cells. J. Cell Sci., 113, Pt. 19: 3409–3417. mutation in the delta-sarcoglycan gene. Nat. Genet., 14: 195–198. Crosbie, R.H., Lebakken, C.S., Holt, K.H., Venzke, D.P., Straub, V., Lee, Noguchi, S., McNally, E.M., Ben Othmane, K., Hagiwara, Y., Mizuno, Y., J.C., Grady, R.M., Chamberlain, J.S., Sanes, J.R., and Campbell, K.P. Yoshida, M., Yamamoto, H., Bonnemann, C.G., Gussoni, E., Denton, 1999. Membrane targeting and stabilization of sarcospan is mediated by P.H., Kyriakides, T., Middleton, L., Hentati, F., Ben Hamida, M., the sarcoglycan subcomplex. J. Cell Biol., 145: 153–165. Nonaka, I., Vance, J.M., Kunkel, L.M., and Ozawa, E. 1995. Mutations Culligan, K.G., Mackey, A.J., Finn, D.M., Maguire, P.B., and Ohlendieck, in the dystrophin-associated protein gamma-sarcoglycan in chromosome K. 1998. Role of dystrophin isoforms and associated proteins in mus- 13 muscular dystrophy. Science, 270: 819–822. cular dystrophy (review). Int. J. Mol. Med., 2: 639–648. Noguchi, S., Wakabayashi, E., Imamura, M., Yoshida, M., and Ozawa, E. Dalloz, C., Claudepierre, T., Rodius, F., Mornet, D., Sahel, J., and Rendon, 1999. Developmental expression of sarcoglycan gene products in A. 2001. Differential distribution of the members of the dystrophin cultured myocytes. Biochem. Biophys. Res. Commun., 262: 88–93. glycoprotein complex in mouse retina: effect of the mdx(3Cv) mutation. O’Kane, C.J. 2003. Modelling human diseases in Drosophila and Mol. Cell. Neurosci., 17: 908–920. Caenorhabditis. Semin. Cell Dev. Biol., 14: 3–10. Dekkers, L.C., van der Plas, M.C., van Loenen, P.B., den Dunnen, J.T., van Ozawa, E., Mizuno, Y., Hagiwara, Y., Sasaoka, T., and Yoshida, M. 2005. Ommen, G.J., Fradkin, L.G., and Noordermeer, J.N. 2004. Embryonic Molecular and cell biology of the sarcoglycan complex. Muscle Nerve, expression patterns of the Drosophila dystrophin-associated glyco- 32: 563–576. protein complex orthologs. Gene Expr. Patterns, 4: 153–159. Radojevic, V., Lin, S., and Burgunder, J.M. 2000. Differential expression Durbeej, M. and Campbell, K.P. 2002. Muscular dystrophies involving of dystrophin, utrophin, and dystrophin-associated proteins in human the dystrophin-glycoprotein complex: an overview of current mouse muscle culture. Cell Tissue Res., 300: 447–457. models. Curr. Opin. Genet. Dev., 12: 349–361. Ramirez-Sanchez, I., Rosas-Vargas, H., Ceballos-Reyes, G., Salamanca, Durbeej, M., Cohn, R.D., Hrstka, R.F., Moore, S.A., Allamand, V., F., and Coral-Vazquez, R.M. 2005. Expression analysis of the SG- Davidson, B.L., Williamson, R.A., and Campbell, K.P. 2000. Disrup- SSPN complex in smooth muscle and endothelial cells of human umbili- tion of the beta-sarcoglycan gene reveals pathogenetic complexity of cal cord vessels. J. Vasc. Res., 42: 1–7. limb-girdle muscular dystrophy type 2E. Mol. Cell, 5: 141–151. Roberds, S.L., Leturcq, F., Allamand, V., Piccolo, F., Jeanpierre, M., Ettinger, A.J., Feng, G., and Sanes, J.R. 1997. epsilon-Sarcoglycan, a Anderson, R.D., Lim, L.E., Lee, J.C., Tome, F.M., Romero, N.B., and et broadly expressed homologue of the gene mutated in limb-girdle mus- al. 1994. Missense mutations in the adhalin gene linked to autosomal cular dystrophy 2D. J. Biol. Chem., 272: 32534–32538. recessive muscular dystrophy. Cell, 78: 625–633. Fort, P., Estrada, F.J., Bordais, A., Mornet, D., Sahel, J.A., Picaud, S., Sandona, D., Gastaldello, S., Martinello, T., and Betto, R. 2004. Charac- Vargas, H.R., Coral-Vazquez, R.M., and Rendon, A. 2005. The terization of the ATP-hydrolysing activity of alpha-sarcoglycan. sarcoglycan-sarcospan complex localization in mouse retina is indepen- Biochem. J., 381: 105–112.

179 R. Hashimoto and M. Yamaguchi

Sekiguchi, M. 2005. The role of dystrophin in the central nervous system: in the six autosomal recessive limb-girdle muscular dystrophies. Hum. a mini review. Acta Myol., 24: 93–97. Mol. Genet., 5: 1963–1969. Shi, W., Chen, Z., Schottenfeld, J., Stahl, R.C., Kunkel, L.M., and Chan, Wheeler, M.T., Zarnegar, S., and McNally, E.M. 2002. Zeta-sarcoglycan, Y.M. 2004. Specific assembly pathway of sarcoglycans is dependent on a novel component of the sarcoglycan complex, is reduced in muscular beta- and delta-sarcoglycan. Muscle Nerve, 29: 409–419. dystrophy. Hum. Mol. Genet., 11: 2147–2154. Signas, C., Katze, M.G., Persson, H., and Philipson, L. 1982. An adeno- Zimprich, A., Grabowski, M., Asmus, F., Naumann, M., Berg, D., virus glycoprotein binds heavy chains of class I transplantation Bertram, M., Scheidtmann, K., Kern, P., Winkelmann, J., Muller- antigens from man and mouse. Nature, 299: 175–178. Myhsok, B., Riedel, L., Bauer, M., Muller, T., Castro, M., Meitinger, T., Straub, V., Duclos, F., Venzke, D.P., Lee, J.C., Cutshall, S., Leveille, C.J., Strom, T.M., and Gasser, T. 2001. Mutations in the gene encoding and Campbell, K.P. 1998. Molecular pathogenesis of muscle degenera- epsilon-sarcoglycan cause myoclonus-dystonia syndrome. Nat. Genet., tion in the delta-sarcoglycan-deficient hamster. Am. J. Pathol., 153: 29: 66–69. 1623–1630. Vainzof, M., Passos-Bueno, M.R., Canovas, M., Moreira, E.S., Pavanello, (Received for publication, September 15, 2006 R.C., Marie, S.K., Anderson, L.V., Bonnemann, C.G., McNally, E.M., and accepted, November 23, 2006) Nigro, V., Kunkel, L.M., and Zatz, M. 1996. The sarcoglycan complex

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