Chie Matsuda

Human PTRF mutations cause secondary deficiency of caveolins resulting in muscular dystrophy with generalized lipodystrophy

Caveolae are invaginations of the plasma membrane involved in many cellular processes, including clathrin-independent endocytosis, cholesterol transport, and signal transduction. They are characterized by the presence of caveolin proteins. Mutations that cause deficiency in caveolin-3, which is expressed exclusively in skeletal and cardiac muscle, have been linked to muscular dystrophy. Polymerase I and transcript release factor (PTRF; also known as cavin) is a caveolar-associated protein suggested to play an essential role in the formation of caveolae and the stabilization of caveolins. Here, we identified PTRF mutations in 5 nonconsanguineous patients who presented with both generalized lipodystrophy and muscular dystrophy. Muscle hypertrophy, muscle mounding, mild metabolic complications, and elevated serum creatine kinase levels were observed in these patients. Skeletal muscle biopsies revealed chronic dystrophic changes, deficiency and mislocalization of all 3 caveolin family members, and reduction of caveolae structure. We generated expression constructs recapitulating the human mutations; upon overexpression in myoblasts, these mutations resulted in PTRF mislocalization and disrupted physical interaction with caveolins. Our data confirm that PTRF is essential for formation of caveolae and proper localization of caveolins in human cells and suggest that clinical features observed in the patients with PTRF mutations are associated with a secondary deficiency of caveolins.

Distal anterior compartment myopathy : A dysferlin mutation causing a new muscular dystrophy phenotype

We report a family with a new phenotype of autosomal recessive muscle dystrophy caused by a dysferlin mutation. The onset of the illness is distal, in the muscles of the anterior compartment group. The disease is rapidly progressive, leading to severe proximal weakness. Muscle biopsy showed moderate dystrophic changes with no vacuoles. Dysferlin immunostaining was negative. Gene analysis revealed a frameshift mutation in the exon 50 (delG5966) of the DYSF gene. This phenotype further demonstrates the clinical heterogeneity of the dysferlinopathies. Ann Neurol 2001;49:130–134

Augmented autocrine bone morphogenic protein (BMP) 7 signaling increases the metastatic potential of mouse breast cancer cells

As malignant breast cancers progress, they acquire the ability to spread to other regions of the body, including bone and lung, but the molecular mechanism underlying the increase in metastatic potential is not fully understood. Here we studied murine 4T1E/M3 highly bone marrow metastatic breast cancer cells, which we established previously. These cells show upregulated expression of bone morphogenetic protein (BMP) 7 and BMP receptors, as well as augmented phosphorylation of Smad1/5/8. Both anchorage-independent cell growth measured in colony forming assays and cell migration measured in wound healing assays were suppressed in 4T1E/M3 cells following treatment with a neutralizing anti-BMP7 antibody or knockdown of BMP7 gene expression. In addition, metastasis of 4T1E/M3 cells to the spine and lung and intracellular levels of phosphorylated Smad1/5/8 were suppressed by knocking down BMP7. Conversely, overexpression of BMP7 in the weakly metastatic parental 4T1E cells augmented their anchorage-independent growth, migration and metastasis to spine and lung. Taken together, our results strongly suggest that augmented autocrine BMP7 signaling leads to increases in the anchorage-independent cell growth, migration and metastatic potential in our bone marrow metastatic breast cancer model.

The γ-Parvin-Integrin-Linked Kinase Complex Is Critically Involved in Leukocyte-Substrate Interaction

Leukocyte extravasation is an important step of inflammation, in which integrins have been demonstrated to play an essential role by mediating the interaction of leukocytes with the vascular endothelium and the subendothelial extracellular matrix. Previously, we identified an integrin-linked kinase (ILK)-binding protein affixin (β-parvin), which links initial integrin signals to rapid actin reorganization, and thus plays critical roles in fibroblast migration. In this study, we demonstrate that γ-parvin, one of three mammalian parvin family members, is specifically expressed in several lymphoid and monocytic cell lines in a complementary manner to affixin. Like affixin, γ-parvin directly associates with ILK through its CH2 domain and colocalizes with ILK at focal adhesions as well as the leading edge of PMA-stimulated U937 cells plated on fibronectin. The overexpression of the C-terminal fragment containing CH2 domain or the depletion of γ-parvin by RNA interference inhibits the substrate adhesion of MCP-1-stimulated U937 cells and the spreading of PMA-stimulated U937 cells on fibronectin. Interestingly, the overexpression of the CH2 fragment or the γ-parvin RNA interference also disrupts the asymmetric distribution of PTEN and F-actin observed at the very early stage of cell spreading, suggesting that the ILK-γ-parvin complex is essential for the establishment of cell polarity required for leukocyte migration. Taken together with the results that γ-parvin could form a complex with some important cytoskeletal proteins, such as αPIX, α-actinin, and paxillin as demonstrated for affixin and actopaxin (α-parvin), the results in this study suggest that the ILK-γ-parvin complex is critically involved in the initial integrin signaling for leukocyte migration.

Tissue-specific isoforms of the bovine mitochondrial ATP synthase γ-subunit

Tissue‐specific isoforms of the γ‐subunit of the bovine F0F1‐ATP synthase were identified for the first time. The two isoforms, heart and liver type, were generated by alternative splicing, the liver‐type RNA transcript containing a 37‐nucleotide sequence as a cassette exon. Protein sequencing of the C‐terminal fragments of the two isoforms of the F1γ‐subunit indicated that the liver‐type isoform had an additional aspartate residue at the C terminus, not present in the heart type one.

The C2A domain in dysferlin is important for association with MG53 (TRIM72)

In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild-type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP-tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair.

Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.

Myotilin is a muscle-specific Z disk protein. Several missense mutations in the myotilin gene (MYOT) have been identified in limb girdle muscular dystrophy (LGMD), myofibrillar myopathy, and distal myopathy patients. All previously reported pathogenic MYOT mutations have been identified only in Exon 2. We sequenced MYOT in 138 patients diagnosed as having LGMD, myofibrillar myopathy, or distal myopathy, and identified a novel MYOT mutation in Exon 9 encoding the second immunoglobulin-like domain in 1 patient with clinically typical LGMD. By light microscopy, there were scattered fibers with rimmed vacuoles and myofibrillary disorganization in the patients muscle biopsy; accumulation of Z disk proteins was observed by immunohistochemistry. Immunoblot analysis demonstrated that the amount of myotilin monomer was increased in the patient muscle, but that the myotilin homodimeric band was decreased. Functional analysis of the myotilin mutation using a yeast 2-hybrid system revealed defective homodimerization of the mutant myotilin and decreased interaction between mutant myotilin and &agr;-actinin. The homodimerization defect was further demonstrated by immunoprecipitation. This is the first MYOT mutation outside of Exon 2 in an LGMD type 1A patient and the first MYOT mutation identified in the Japanese population. This mutation in the second immunoglobulin-like domain impairs myotilin dimerization and alters the binding between myotilin and &agr;-actinin, which is known to be important for actin bundling.

Affixin activates Rac1 via βPIX in C2C12 myoblast

— MINT‐6179203, MINT‐6179212, MINT‐6178859, MINT‐6178812, MINT‐6178832, MINT‐6178843: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by coimmunoprecipitation (MI:0019) — MINT‐6179221: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with αpix (uniprotkb:Q8K4I3) by coimmunoprecipitation (MI:0019) — MINT‐6178962, MINT‐6178983: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179002, MINT‐6179021: Affixin (uniprotkb:Q9HBI1) binds (MI:0407) βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179039: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by pull-down (MI:0096) — MINT‐6179054: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Cdc42 (uniprotkb:P70766) by pull-down (MI:0096) — MINT‐6178790: Affixin (uniprotkb:Q9HBI1) and αpix (uniprotkb:Q8K4I3) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178760: Affixin (uniprotkb:Q9HBI1) and βpix (uniprotkb:Q9ES28) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178801: Affixin (uniprotkb:Q9HBI1) and dysferlin (uniprotkb:Q9ESD7) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178779: Affixin (uniprotkb:Q9HBI1) and ILK (uniprotkb:O55222) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin sjl/sjl mice to the fukutin-knock-in fukutin Hp/− and Large-deficient Large myd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin Hp/− mice do not show a dystrophic phenotype; however, (dysferlin sjl/sjl: fukutin Hp/−) mice showed a deteriorated phenotype compared with (dysferlin sjl/sjl: fukutin Hp/+) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin Hp/− mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin Hp/− FCMD mouse model, and the (dysferlin sjl/sjl: fukutin Hp/−) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.

Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair.

Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy. Objectives: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy. Methods: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts. Results: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation. Conclusion: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.