41⁰ Encontro Anual da Sociedade Americana de Biologia Celular - Resumos

1) Dystrophin delivery in mdx mice via retrovirally transduced early myogenic progenitor cells

Makoto Ikezawa¹, Hairong Peng², Zhuqing Qu², Xiao Xiao³, Ryan Pruchnic², Baohong Cao¹, Teruhisa Miike⁴, Johnny Huard², ¹Growth and Development Lab., Children's Hospital of Pittsburgh, Rm 4151 Rangos Research center, 3460 Fifth Ave., Pittsburgh, PA 15213, ²Department of Orthopaedics, University of Pittsburgh, ³Department of Molecular Genetics and Biochemistry, University of Pittsburgh, ⁴Department of Child Development, Kumamoto University School of Medicine, Japan

Duchenne Muscular Dystrophy (DMD) is characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers, which results in the clinical manifestation of progressive muscle weakness. Thus far, two approaches aimed at delivering dystrophin into dystrophic muscle have been investigated: 1) gene therapy and 2) myoblast transplantation. Gene therapy has been limited by immune rejection problems and viral cytotoxicity of virus vectors. Myoblast transplantation has also been hindered by immune rejection problems, in addition to a poor survival rate of the injected myoblasts. We have investigated the use of retroviral vectors to deliver the dystrophin gene through the ex vivo approach using early myogenic progenitor cells. We have recently engineered a retrovirus vector which encodes the human mini-dystrophin gene (RetroDys3999). Early myogenic progenitor cells were isolated from mdx mice (animal model for DMD) by the preplating technique, and transduced by the RetroDys3999. The transduced cells expressed desmin (a myogenic marker), and also the stem cell markers CD34 and Sca-1 in vitro,  suggesting similar characteristics to muscle derived stem cells. Transplantation of the transduced cells can be used to deliver human mini-dystrophin genes into the skeletal muscle of mdx mice. Although an infiltration of CD4 and CD8 activated lymphocytes was detected in the injected cell area, it is unclear whether these lymphocytes were triggered by the injected cells, since the dystrophic pathophysiology also results to infiltration of lymphocytes. Surprisingly, the number of dystrophin positive myofibers at 8 weeks post-injection (55.7 ± 34.7, n=3) was not significantly different from the results obtained at 2 weeks post-injection (65.0 ± 20.3, n=3). The combination of myoblast transfer using early myogenic progenitor cells and gene therapy using retrovirus vectors may help in the development of strategies to achieve a persistent dystrophin delivery which may consequently alleviate the muscle weakness in DMD.

 2) Muscle regeneration delay in tensin knockout mice

Akiko Ishii¹, Norio Ohkoshi², Shin'ichi Shoji², Su Hao Lo³, ¹Neurology, Tsukuba So-ai Hospital, 1008 Takasaki, Kukizaki, Inashiki-Gun, 300-1245 Japan, ²Neurology, University of Tsukuba, Japan, ³Orthopaedic, University of California, Davis

Tensin is an actin binding protein that is important for the regulation of actin assembly at focal adhesions. Previous reports suggest that tensin not only maintain cell structure but is also involved in signal transduction. We had reported that the muscle regeneration process induced by cardiotoxin lasted longer in tensin knockout mice than in wild-type mice. Withdrawal from cell cycle and fusion capacity was less in isolated knockout myoblasts in vitro. To determine which process of muscle generation was impaired in tensin knockout mice in vivo, the anterior tibial muscles were isolated and protein and RNA were extracted from them at 1, 3, 5, 7, 14, 28, and 56 days after caidiotoxin injection. The expression of dystrophin-associated proteins and nNOS were delayed but beta-dystroglycan and  caveolin were not. The RT-PCR of myoD showed delayed expression in tensin knockout mice. Our results demonstrated that tensin may play a role in muscle regeneration process, especially satellite cell activation and fusion.

3) Dystrophin and Utrophin are Functionally Homologous Actin Binding Proteins But Act Through Distinct Modes of Filament Association

Inna N. Rybakova¹, Jitandrakumar R. Patel¹, Kay E. Davies², Peter D. Yurchenco³, James M. Ervasti¹, ¹Dept. of Physiology, University of Wisconsin, ²Dept. of Human Anatomy and Genetics, University of Oxford, United Kingdom, ³Dept. of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School

Dystrophin and utrophin are homologous proteins thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma.  While utrophin overexpression corrects several phenotypic parameters associated with dystrophin deficiency, recent studies question whether utrophin can fully replace the actin binding functions normally provided by dystrophin.  Here, we demonstrate that utrophin overexpression in dystrophin-deficient mdx muscle restores strong mechanical coupling between costameric actin and the sarcolemma.  We have also expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers.  Contrary to predictions based on studies of isolated protein fragments, we demonstrate that utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization.  However, utrophin appears to bind laterally along actin filaments through acidic spectrin-like repeats rather than the cluster of basic repeats employed by dystrophin.  Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but also suggest that the molecular epitopes important for filament binding differ between the two proteins.  Supported by NIH grants AR42423 and AR01985 (J.M.E.), the Muscular Dystrophy Association (I.N.R.) and the Medical Research Council, UK (K.E.D.).

4) A Chronic Inflammatory Response Dominates the Skeletal Muscle Molecular Signature in Dystrophin-Deficient mdx Mice

John D Porter¹, Sangeeta Khanna², Henry J Kaminski³, J Sunil Rao⁴, Anita P Merriam², Chelliah R Richmonds³, Patrick Leahy⁵, Jingjin Li⁴, Francisco H Andrade³, ¹Ophthalmology, Case Western Reserve University and University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-5068, ²Ophthalmology, Case Western Reserve University, ³Neurology, Case Western Reserve University, ⁴Epidemiology and Biostatistics, Case Western Reserve University, ⁵Comprehensive Cancer Center, Case Western Reserve University

Mutations in dystrophin cause Duchenne muscular dystrophy (DMD), but absent dystrophin does not necessarily produce necrosis in all muscles, life stages, and species.  Using DNA microarray in mdx vs. control mice, we screened ~10,000 genes to establish a broad molecular signature of dystrophinopathy.  Data provide evidence that secondary mechanisms are key, if not essential, contributors to pathogenesis.  Our experimental strategy used variability controls, adequate replicates (5 arrays/group), and stringent analytic tools, including Significance Analysis of Microarrays to estimate and manage false positive rates.  This conservative approach identified 249 differentially expressed genes (212 known/37 ESTs) in mdx hindlimb, > 75% of which have not been previously reported in human or animal dystrophies.  A muscle group that is spared from dystrophic changes, extraocular muscle, did not exhibit these alterations in gene expression.  Data provide detailed evidence for all phases of inflammation in mdx (30% of differentially expressed genes), including cytokine and chemokine signaling, leukocyte adhesion and diapedesis, invasive cell type-specific markers, and complement system activation.  Many of these genes are potential therapeutic targets, either for primary treatment of DMD or in adjuvant therapy to improve efficacy of myoblast transplantation.  Our data also identified a novel signaling link between mononuclear cells and repair processes.  Up-regulation of extracellular matrix mRNA in mdx was nearly identical to that previously reported in DMD, although mdx does not exhibit the severe fibrosis characteristic of DMD.  The gene profiling approach establishes that disease gene products can no longer be seen only in simple relationships with their local networks of interacting proteins, particularly when a multidimensional protein like dystrophin is associated with a complex disease like muscular dystrophy.

5) Spontaneous fusion of bone marrow cells with embryonic stem cells generates pluripotent stem cells

Naohiro Terada¹, Takashi Hamazaki¹, Masahiro Oka¹, Masanori Hoki¹, Yuka Nakano², Edwin M Meyer², Laurence Morel¹, Bryon E Petersen¹, Edward W Scott³, ¹Pathology, U. Florida, P. O. Box 100275, Gainesville, FL 32610, ²Pharmacology, U. Florida, ³Shands Cancer Center, U. Florida

Here we demonstrate that mouse bone marrow cells can fuse spontaneously with other cells and subsequently adopt the phenotype of the donor cells. A simple co-culture of bone marrow cells from female green fluorescent protein (GFP)-transgenic mice with embryonic stem (ES) cells from male non-fluorescent mice produced GFP-positive cells morphologically and biochemically resembling to ES cells. The cells maintained in an undifferentiated phenotype when cultured in a medium containing leukemia inhibitory factor (LIF), and differentiated into all three germ layer cells in vitro upon removal of LIF. Of interest, the cells had a tetraploid XXXY karyotype, and a hybrid genotype of bone marrow and ES cells, indicating that spontaneous cell fusion had occurred during the co-culture. The data indicate that the pluripotent phenotype of ES cells can be dominant upon cell fusion with primary bone marrow cells. Moreover, considering recent reports of transplanted bone marrow cells turning into unexpected cell types in vivo, the present data raise a warning to the feverish trend in stem cell research to conclude transdifferentiation or dedifferentiation of cells in vivo without careful examination of their genotypes.

6) Muscle derived stem cells contribute to blood vessel and peripheral nerve formation

zhuqing Qu-Petersen, John Mytinger, Johnny Huard, Growth and Development Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, 4151 Rangos Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213

Although muscle derived stem cells (MDSCs) isolated from different groups have shown the capability of generating muscle, blood and osteogenic cells, their ability to differentiate into more lineages is still unclear. In the present study, we investigate the possibility of pluripotential differentiation of MDSCs into peripheral nerve and blood vessels after injection in skeletal muscle. Primary muscle cultures were prepared from newborn normal mice, and the MDSCs were clonally isolated from the primary culture by a modified preplate technique.  The MDSCs were transduced with retrovirus carrying nuclei LacZ reporter gene, and injected into the m. gastrocnemius of mdx mice (an animal model for Duchenne Muscular Dystrophy). The animals were sacrificed at 10 and 25 days post-injection; the injected muscles were harvested and stained with LacZ and H.E. or co-stained with different antibodies by immunochemistry to reveal the expression of dystrophin (a muscle specific protein), M-Cadherin (a marker of muscle satellite cells), CNPase (a marker of  Schwann cells in the peripheral neural system), and von Willebrand factor (vWF) (a marker of endothelial cells of blood vessels). Results: Large engraftments with many LacZ(+) or dystrophin(+) myofibers were observed in the transplanted muscle, and  LacZ(+)  nuclei were also found in either vascular or peripheral  nerve-like structrues 10 days post-transplantation. Co-localization of LacZ with M-Cadherin, CNPase and vWF demonstrated the ability of injected LTP cells to differentiate into muscle satellite cells, Schwann cells and endothelial cells.  2) By 25 days post-transplantation, many LacZ (+) myofibers were still observed in transplanted muscle, and some LacZ(+) nuclei were found in well-differentiated blood vessels and peripheral nerves. We therefore conclude that MDSCs isolated by preplate technique are pluripotent, which may be useful in cell therapy for both muscle and non-muscle disease.

7) Tissue-Specific Disruption of Dystroglycan:  Insights Into Muscular Dystrophy

Kevin P. Campbell¹, Ronald D. Cohn², Michael D. Henry², Rita Barresi², Fumiaki Saito², Steven A. Moore³, C. Ronald Kahn⁴, Roger Williamson⁵, ¹University of Iowa, HHMI, 400 EMRB, Iowa City, IA 52242, ²University of Iowa, Dept. of Physiology and Biophysics, HHMI, ³Department of Pathology, University of Iowa, ⁴Joslin Diabetes Center, Harvard Medical School, ⁵Department of Obstetrics and Gynecology, University of Iowa

Dystroglycan is a central component of the dystrophin-glycoprotein complex, a complex which has an essential role in a variety of muscular dystrophies.  In order to understand better the function of dystroglycan in skeletal muscle we engineered mice with a specific disruption of dystroglycan in striated muscle (MCK-DG-null mice).  Interestingly, MCK-DG-null mice develop muscular dystrophy and exhibit specific disruption of dystroglycan at the sarcolemma, while neuromuscular junctions and myotendinous junctions show normal levels of dystroglycan expression.  However, when MCK-DG-null mice reach an advanced age no evidence of tissue fibrosis and or fat replacement is observed.  Instead, MCK-DG-null mice maintain their ability to efficiently regenerate skeletal muscle and develop significant skeletal muscle hypertrophy, which is in marked contrast to dystrophin deficient mdx mice and sarcoglycan-null mice.  Here we demonstrate, that dystroglycan is expressed in satellite cells in which Cre is not expressed by the MCK-Cre promoter.  Hence, continuous activation of satellite cells leads to residual expression of dystroglycan in fibers undergoing regeneration.  Importantly, we present the first in vivo evidence that the loss of regenerative capacity is not a function of age in mice undergoing repetitive cycles of degeneration and regeneration during the course of muscular dystrophy.  Moreover, our results demonstrate that maintenance of myogenic differentiation and self-renewing potential of muscle cells is a desirable paradigm for the development of future therapeutic options for muscular dystrophy.

8) The capability of muscle stem cells to restore dystrophin and repopulate ablated bone marrow in mice

Baohong Cao, Shigemin Kimura, Makoto Ikezawa, Jeffrey G Kuremsky, Ryan Pruchnic, James Cummins, Johnny Huard, Dept of Orthopaedic Surgery, University of Pittsburgh

Duchenne muscular dystrophy (DMD) is a congenital muscle disease characterized by a lack of dystrophin expression resulting in early death due to respiratory and cardiac failure. Myoblast transplantation (MT) is a way to deliver dystrophin to the diseased muscle. We have isolated a population of muscle derived cells (MDC) that improve myoblast mediated gene transfer to skeletal muscle. These cells express markers similar to those found on hematopoietic stem cells. The MDC were isolated from dystrophic mdx mice, cloned (MC13) and transfected with a plasmid encoding the LacZ, mini dystrophin and neomycin resistant genes. 0.5 x 106 MC13 cells were injected into the tail vein of adult mdx mice and were sacrificed 7 days post injection. Upon examination of the hindlimb muscles, some myofibers were found co-expressing LacZ and dystrophin. Although the number of LacZ and dystrophin positive myofibers was low (10-20 fibers per section), the ability of these muscle-derived cells to disseminate systemically suggests that these cells may display an increased migratory capacity. Since we observed that some of the injected cells were found in various organs including bone marrow, we have tested whether these cells can reconstitute bone marrow of mdx mice. We injected MC13 cells into lethally irradiated mice and found that the MC13 cells increased the survival of the mice up to 6 months post-transplantation. Immunocytochemistry revealed that the MC13 cells that were recovered from the recipients expressed CD45, a marker specific for cells of hematopoietic lineage and Gr-1, a marker correlated with granulocyte differentiation and maturation. This demonstrated that MC13 cells could differentiate into hematopoietic lineage and therefore prolonged the life span of the lethally irradiated mice. Continued investigation into improving MC13's migratory ability could lead to a major breakthrough for cell therapy in the future

9) Golgi-localisation of fukutin and fukutin-related protein: implications for muscular dystrophy

Derek J Blake¹, Martin Brockington², Francesco Muntoni², Matthew A Benson¹, ¹Human Anatomy and Genetics, Oxford University, South Parks Road, Oxford, OX1 3QX United Kingdom, ²Dubowitz Neuromuscular Centre, Imperial College School of Medicine, United Kingdom

Mutations in the fukutin gene cause Fukuyama congenital muscular dystrophy (FCMD), a severe muscle disease that is also associated with abnormal neuronal migration. Recently, we identified a fukutin-related protein (FKRP) that is mutated in some forms of congenital muscular dystrophy (CMD1C). Sequence analysis of fukutin and FKRP suggests that both proteins are involved in protein modification and are probable phosphoryl-ligand transferases. Both proteins contain a conserved DxD motif that is found in the active sites of many glycosyltransferases.

To gain further insight into the cellular role of fukutin and FKRP, we determined the subcellular localisation of both proteins in cultured cells. Both proteins co-localise with alpha-mannosidase II in the cis- and medial- Golgi apparatus of NRK cells. The Golgi-localisation signal for both proteins was limited to the first 35 N-terminal amino acids that included the transmembrane domain indicating that both proteins possess a Golgi-signal anchor. Both proteins were re-distributed into the cytoplasm of NRK cells following treatment with brefeldin A or nocodazole. In transfected HEK 293T cells over-expressing fukutin or FKRP we were unable to show that either protein was secreted into the media. However, both proteins co-immunoprecipitate from transfected cells suggesting that they physically interact with each other.

Our data suggest that fukutin and FKRP are Golgi-resident proteins. We propose that fukutin and FKRP are involved in the glycosylation of proteins that form the basal lamina in muscle such as α-dystroglycan and laminin. Muscular dystrophy arises in patients lacking these proteins due aberrant protein glycosylation that affects a variety of protein:glycoprotein interactions.

10)The Dystroglycan Complex and Cell Survival Signaling in Muscle Cells

Kurt J Langenbach¹, Thomas A Rando², ¹Department of Neurology/Neurological Sciences, Stanford University, 300 Pasteur Drive Room A-343 Mail Code 5235, Stanford, CA 94305-5235, ²Neurology Service and GRECC, 3801 Miranda Ave, Palo Alto CA, 94304, VAPAHCS

The pathogenetic mechanisms leading to muscle deterioration in muscular dystrophies are relatively unknown. Numerous muscular dystrophies are the result of disruption of proteins comprising the dystrophin glycoprotein complex (DGC). One well established mechanism leading to induction of apoptosis in many cell types is the disruption of cell/ extracellular matrix interactions. Co-localization of proteins involved in signaling, such as FAK and Grb2, with members of the DGC suggests that the DGC may assist in the transmission of signals across the cell membrane. Since apoptosis is an early feature of muscular dystrophy in vivo, we are investigating the role of the DGC in the propagation and maintenance of cell survival signals in muscle cells.  Our working hypothesis is that the cell death found in muscular dystrophies is due to disruption of cell survival pathways mediated by the dystroglycan complex. A blocking antibody against α-dystroglycan (IIH6) was used to disrupt the interaction between α-dystroglycan and the extracellular matrix, and a non-blocking antibody to α-dystroglycan (VIA41) served as a control. Increased TUNEL staining in IIH6 treated cells as compared to controls suggests a role for the DGC in survival signaling. Since phosphorylated AKT has been associated with cell survival signaling downstream of FAK, and pharmacological inhibitors of PI3K decrease AKT phosphorylation and induce apoptosis in muscle cell cultures, we measured the phosphorylation state of AKT and its downstream effectors in cells treated with IIH6. Our results demonstrate a decrease in phosphorylation levels of AKT and its downstream effectors in cells in which the DGC has been disrupted. These results suggest a role for PI3K/AKT in mediating cell survival signals mediated by the DGC, and provide new insight into the molecular pathogenesis associated with the development of muscular dystrophies.

11) The Myotonic Dystrophy Protein Kinase Regulates Membrane/Cytoskeleton Dynamics

Feizhou Liu¹, Rongxin Zhang¹, Shida Jin¹, Miho Shimizu¹, Wenfu Wang¹, Andrea Muranyi², David J Hartshorne², Henry F Epstein¹, ¹Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, ²Muscle Biology Group, University of Arizona

Myotonic dystrophy is a multi-system disease caused by the expansion of the CTG triplet repeats in the 3' untranslated region of the myotonic dystrophy locus. The gene encodes a protein kinase (DMPK) that is homologous to a variety of protein kinases involved in the regulation of membrane/cytoskeleton dynamics. In order to study the molecular mechanism of the disease, DMPK cDNA was transfected into human and rabbit lens cells. The transfected cells showed apoptotic-like membrane blebbing and actin cytoskeleton reorganization, and both events required the kinase activity of DMPK as a mutation in the kinase active site eliminated such cellular changes. Rac-1 and Raf-1, two important components in cell signal transduction pathways, interact physically with DMPK and up-regulate its kinase activity. The myosin phosphatase dephosphorylates the regulatory light chain of myosin II, thus inhibiting actomyosin contractility and, by doing so, regulates many cellular events. Recombinant DMPK, like Rho kinase, phosphorylated the same threonine residue of MYPT1, the substrate targeting subunit of myosin phosphatase, and consequently inhibited myosin phosphatase activity. In contrast to Rho kinase, DMPK did not directly phosphorylate the regulatory myosin light chain to activate actomyosin activity, and its phosphorylation of MYPT1 was not inhibited by Y27632. We propose that DMPK may regulate membrane/cytoskeleton dynamics in the cell through myosin phosphatase and in collaboration with other proteins including Rho kinase and myosin light chain kinase, and this regulation is a response to cellular signaling relayed through Rac-1 and Raf-1. Supported by NIH grants NS 35071 (to HFE) and HL 23615 (to DJH) and an MDA grant (to HFE).  

12) Caveolin-3 mutations disrupt ERK 1/2 signaling in skeletal myotubes in vitro

Gayle M Smythe¹, Josh Eby¹, Bethanne Deuel¹, Marie-Helene Disatnik¹, Thomas A Rando², ¹Department of Neurology and Neurological Science, Stanford University, 300 Pasteur Drive, Mail Code 5235, Stanford, CA 94305-5235, ²Neurology Service and GRECC, 3801 Miranda Ave, Palo Alto CA, 94304, VAPAHCS

Dominant negative mutations in the caveolin-3 gene that cause a deficiency of caveolin-3 from the sarcolemma of skeletal muscle fibers, are responsible for human limb girdle muscular dystrophy type 1C.  Caveolin-3 is a muscle-specific isoform of the caveolin family, which are a major protein component of invaginations in the plasma membrane termed caveolae.  Caveolins are characterized by a "scaffolding domain" that binds several families of receptor and non-receptor protein kinases, and therefore are likely to have an essential regulatory role in many intracellular signaling cascades.  Perturbations in signaling pathways may contribute to the susceptibility of skeletal muscle to degeneration that is characteristic of the muscular dystrophies.  The aim of this study was to determine if a deficiency of caveolin-3 at the sarcolemma of skeletal myotubes (caused by the expression of mutant caveolin-3 cDNA) would disrupt intracellular signaling.  Skeletal myoblasts transfected with mutant caveolin-3 cDNA were induced to undergo differentiation and fusion.  All cells fused to form multinucleated myotubes, although cells harboring mutant caveolin-3 were more susceptible to loss of cell-matrix adhesion in longer term (up to 7 days) culture.  Phosphorylation of extracellular signal-related kinase 1/2 (ERK 1/2) was downregulated in cells harboring caveolin-3 mutants, compared with normal controls.  This is likely to be effected upstream by changes in Ras signaling, as Ras is known to associate with the caveolin scaffolding domain, and in Grb2/Shc complex formation.  These results demonstrate that the absence of caveolin-3 at the cell membrane disrupts ERK 1/2 signaling in skeletal muscle cells in vitro.  Disrupted intracellular signaling pathways caused by a deficiency in certain proteins at the sarcolemma may provide new targets for pharmacological intervention in muscle weakness and degeneration in muscular dystrophies.

13) Ubiquilin-1 binds to myotonic dystrophy protein kinase (DMPK) and modulates its kinase activity and degradation

Shinichiro Nagamitsu¹, Gyorgyi Horvath¹, Jose M Barral², Feizhou Liu², Rongxin Zhang², Henry F Epstein², Tetsuo Ashizawa¹, ¹Department of Neurology, Baylor College of Medicine, VAMC 2002 Holcombe Bldg 100 2B-172, Houston, TX 77030, ²Department of Biochemistry, Baylor College of Medicine

An expansion of the CTG repeat in the 3'untranslated region of the DMPK gene causes myotonic dystrophy type 1. DMPK belongs to a group of serine-threonine protein kinases designed as the gmyotonic dystrophy family of protein kinase and has been shown to phosphorylate dihydropyridine receptor, phospholemman, and myosin phosphatase subunit-1 (MYPT-1).  Although DMPK appears to regulate cell size and shape and modulate the Ca²⁺ homeostasis of skeletal muscle, the exact physiological functions of this kinase remain to be elucidated.  In this study, we used the full-length DMPK as the bait in the yeast two-hybrid screening, and identified an ubiquitin-like protein, ubiquilin-1, as an interacting protein.  Ubiquilin-1 shows 99% homology to hPLIC1, which has been postulated to provide a link between the ubiquitination machinery and the proteasome. In vitro binding assay confirmed the interaction, and demonstrated that the Stil-like repeat element domain of ubiquilin-1 is required to bind to the kinase domain of DMPK.  The interaction was also demonstrated in the transfected mammalian cell.  Colocalization of both proteins was immunohistologically demonstrated in the nucleus and the cytoplasm of HEK293 cells. DMPK did not phosphorylate ubiquilin-1; however, the equimolar or a lower amount of ubiquilin-1 (0.5-50nM) increased the kinase activity of DMPK two to four-fold, whereas higher concentrations (0.5-2.0μM) inhibited the DMPK transphosphorylation of a small MYPT-1 peptide. The half-life of DMPK determined by pulse-chase experiments using ³⁵S-methionine was ~43 hours, which was prolonged to ~74 hours by co-transfection of ubiquilin-1, suggesting that ubiquilin-1 inhibits the degradation of DMPK. Our results indicate that ubiquilin-1 interacts with DMPK to modulate its kinase activity and degradation.

 This study was supported by grants from the VA (TA), NINDS (HFE), MDA (HFE), and the Hunter Research Fund (TA & HFE) and a donation from Mr. and Ms. Vasku.

 14)A Role for Lamins in Regulating Nuclear Size

Reynold I Lopez-Soler¹, Timothy P Spann¹, Robert D Moir¹, Robert D. Goldman², ¹Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave, Chicago, IL 60611, ²Northwestern University Medical School

The processes driving the growth of the nucleus during interphase are not well understood.  Based on our recent findings that nuclear lamins play an essential role in the initial assembly of the nuclear envelope, we have investigated whether the ongoing assembly of nuclear lamins is required for the nucleus to increase in size.  We have identified a 100 aa polypeptide derived from the C-terminal domain of lamin B3 (LB3T100), that blocks the incorporation of wild type lamins into the lamina of the assembled nuclei.  Furthermore, LB3T100 prevents the typical increases in nuclear size that are observed in Xenopus extracts. Although LB3T100 inhibited the growth of the nucleus, LB3T100 did not block nuclear transport or the ability of nuclei to exclude large macromolecules (e.g. 70 Kd dextran). However, LB3T100 does block lamin polymerization in vitro.  These results suggest that during interphase normal increases in nuclear size require ongoing assembly of nuclear lamins.  The inhibitory effects on lamin assembly in vitro are no longer observed when a single amino acid in LB3T100 is altered to mimic a lamin A mutation (R454W) associated with Emery-Dreifuss Muscular Dystrophy.  In addition, LB3T100-R454W does not inhibit either normal increases in nuclear size or the incorporation of lamins into the lamina.  Our results suggest that the human diseases associated with mutations in the C-terminal domain of lamins may result from alterations in the normal assembly properties of nuclear lamins. Supported by NCI.

15) Immunoaffinity Purification of the Calpains

Jinyang Cong, Valery F. Thompson, Darrel E. Goll, Muscle Biology Group, University of Arizona

The calpains,  μ- and m-calpain, have received increasing interest because of their  role in a variety of pathological conditions ranging from the muscular dystrophies to damage in ischemic areas near a myocardial infarct or stroke. It has been difficult to assemble the two polypeptide subunits of the calpains from cDNA expression systems into proteolytically active molecules. Therefore, it has been necessary to rely on purification of the calpains from tissues.  Purification of the calpains also is difficult and requires four to six successive column chromatographic steps. Even then, μ-calpain especially, may be only partly pure depending on the tissue source.  Developing improved  purification protocols for the calpains has been hindered because:1) the presence of calcium in the preparations results in rapid autolytic degradation of the calpains, thereby eliminating the possibility of using calcium affinity approaches; and 2) the calpains are easily denatured  and proteolytically active molecules cannot be regenerated after denaturation.  We have found that proteolytically active calpains can be eluted at pH 9.0-9.5 from an immunoaffinity column containing a monoclonal antibody to the small subunit that is common to both μ- and m-calpain. Evidently, binding of the antibody, whose epitope is between amino acids 92-104 on the small subunit, is so weak that the calpains can be eluted under mild conditions. Possibly because of the weak binding, the eluted calpains are purest if the initial tissue extracts are partly purified by successive chromatography on  phenyl Sepharose and DEAE-ion-exchange columns before being loaded onto the immunoaffinity column, although it is possible to obtain preparations that are greater than 70% pure by loading crude extracts directly onto the immunoaffinity columns. Supported by the National Research Initiative Competitive Grants and the Muscular Dystrophy Association. 

16) Transcriptional Profile of Rat Extraocular Muscle by Serial Analysis of Gene Expression (SAGE)

Georgiana Cheng, John D Porter, Ophthalmology, Case Western Reserve University and University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-5068

Extraocular muscle (EOM) exhibits fundamental differences from other skeletal muscle (Porter et al., Proc. Nat. Acad. Sci., in press).  Such muscle group phenotypic differences are invaluable in understanding the mechanisms behind the differential involvement in disease, such as the dramatic EOM sparing in muscular dystrophy.  Yet, because of the highly specialized niche for EOM, it is largely uncharacterized by molecular biology.  Taking advantage of high-throughput sequencing technology, we have used SAGE to generate a quantitative profile of gene expression in adult rat EOM.  54,764 expressed sequence tags were analyzed from EOM polyadenylated RNA; 17,602 of these were unique tags, each potentially representing a different mRNA species. Of these unique tags: (1) 7.8% were detected at high and intermediate levels (tag copies >= 5) and 19.3% were detected at lower levels (tag copies 4 to 2), whereas 72.9% were present as single copy; (2) 40% of the tags matched known expressed sequences, 85.7% of which were matched to one known expressed sequence and 14.3% were matched to more than one known expressed sequence; (3) 60% of the tags had no matches (representing potentially novel genes).  Genes represented by SAGE tags with >= 2 copies were assigned to functional classes. Tag frequencies serve as indices of mRNA relative abundance.  Many of the genes expressed at levels > 0.2% of total tag number are involved in sarcomeric structure, excitation-contraction coupling, intermediary metabolism, and gene/protein expression.  The expression of some genes has not been previously linked to skeletal muscle.  Overall, these data provide a distinctive molecular signature of EOM that will facilitate understanding of developmental regulation of the EOM phenotype and of the mechanisms behind the preferential targeting or sparing of EOM in disorders such as the muscular dystrophies.

17) Caveolin-3 knock-out mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities

Ferruccio Galbiati¹, Daniela Volonte'¹, Michael P. Lisanti², ¹University of Pittsburgh, ²Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells.  Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an ~95 % reduction in caveolin-3 protein expression, i.e., a caveolin-3 deficiency.  Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency.  We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from  lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally-oriented T-tubules.   These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level.

18) Electron Microscopy and 3-D Reconstruction of F-Actin-Dystrophin and F-Actin-Utrophin

William Lehman¹, Victoria Hatch¹, John Kendrick-Jones², Roger Craig³, Andrew Sutherland-Smith², ¹Dept. Physiology & Biophysics, Boston Univ. Sch. Med., 715 Albany St., Boston, MA 02118, ²MRC Laboratory of Molecular Biology, United Kingdom, ³Dept. Cell Biol., Univ. Mass. Med. Sch.

Utrophin (UTR) and dystrophin (DYS) both link the F-actin (AC) cortical cytoskeleton to the cell membrane. UTR is found in developing muscle and non-muscle cells whereas closely related DYS is present in differentiated muscle and brain. DYS, defective in Duchenne and Becker muscular dystrophy, is thought to act as a cellular shock-absorber limiting damage to the sarcolemma during muscle contraction and extension, and it may display distinct structural features essential for its function.  N-terminal AC-binding constructs of UTR and DYS both consist of a pair of CH-domains (CH1-CH2) which crystallize as anti-parallel dimers (ACH1-BCH2/BCH1-ACH2; Keep et al., 1999; Norwood et al., 2000;); however, in vitro, UTR and DYS CH-domain constructs form 1:1 and 2:1 complexes with F-actin, respectively, suggesting that only DYS is dimeric on filaments (ibid.).  In the current study, negative staining and 3-D reconstruction of AC decorated with constructs of either UTR or DYS clearly revealed DYS and UTR densities on AC. Both UTR and DYS bound to the upper surface of AC-subdomain-1. The UTR density formed a projection that lay over subdomain-2 and extended radially toward subdomains-3 and 4, thus corresponding to the structure observed for monomeric fimbrin and calponin CH-domains (Hanein et al. 1997; Hodgkinson, 1997). In fact, the atomic structure of fimbrin CH1-CH2 fitted very well into the UTR density.  In contrast, the DYS density differed qualitatively from those of the other CH-domain constructs, forming a bi-lobed extension. One lobe was similar in shape and orientation to that of the UTR CH1-CH2 and the other apparently represented the second pair of CH-domains seen in the crystal structure of the DYS-dimer. DYS appears to make more extensive contact with AC, an arrangement well suited for its function.

19) Two-hybrid Screen for Novel Lamin A/C Binding Partners from Human Skeletal Muscle

Michael S. Zastrow, Katherine L. Wilson, Cell Biology, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205

The nuclear lamina is a network of filaments that underlie the inner nuclear membrane and may also extend into the nucleus.  The major components of the lamina are nuclear-specific intermediate filament proteins named lamins.  Lamins are essential for viability, and mediate the shape and stability of the nucleus, DNA replication and the spacing of nuclear pore complexes.  Dominant mutations in the gene encoding A-type lamins are associated with at least three diseases: Emery-Dreifuss muscular dystrophy (EDMD), Dunnigan type familial partial lipodystrophy (FPLD) and dilated cardiomyopathy 1C (DC).  Each disease affects a specific tissues: heart, skeletal muscle and tendons (EDMD), adipose (FPLD) and heart (DC).  Based on this specificity we hypothesize that A-type lamins provide attachment sites for one or more transcriptional regulators or other factors important for cardiac, adipose or skeletal muscle function.  To test this hypothesis, we used the yeast two-hybrid method to screen a skeletal muscle library for new binding partners for lamins A/C.  We isolated several putative new binding partners which are currently being verified for lamin A binding and tested for their ability to bind to mutant lamin A proteins.  Results from these experiments will be presented at the meeting.

20) Effects of Autolysis on Properties of the Calpains

Hongqi Li¹, Valery F. Thompson¹, Geert H. Geesink², Merin L. Boehm³, Darrel E. Goll¹, ¹Muscle Biology Group, University of Arizona, ²Institute for Animal Health and Science, University of Arizona, Netherlands, ³Pfizer, Inc.

Ever since it was discovered that the calpains autolyze rapidly when incubated with calcium, the physiological significance of this autolysis has been controversial. The suggestion that autolysis converted a "proenzyme calpain" to an active protease similar to the activation of prothrombin was discounted when it was shown that the unautolyzed calpains are catalytically active proteases. We have compared properities of the autolyzed and unautolyzed calpains to learn whether this comparison would provide clues about the role of autolysis. The autolyzed calpains are more hydrophobic than the unautolyzed calpains. Whereas the unautolyzed calpains can be eluted from phenyl Sepharose hydrophobic columns at very low ionic-strength, the autolyzed calpains require SDS to elute them from phenyl Sepharose. The autolyzed calpains aggregrate in the presence of salt; the aggregated calpains are proteolytically inactive.  The autolyzed calpains lose nearly 75% of their activity within 5 min in 500 mM NaCl or KCl. This loss in activity is more pronounced for autolyzed  μ- than for autolyzed m-calpain and cannot be reversed by dialysis overnight against a low-ionic-strength buffer. Incubation in 500 mM KCl for 45 min has little effect on proteolytic activity of the unautolyzed calpains. HPLC size-exclusion chromatography shows that 500 mM NaCl produces two peaks of autolyzed μ-calpain; a proteolytically inactive peak that elutes before the proteolytically active peak and that presumably is an aggregated species of  μ-calpain.   HPLC size-exclusion chromatography shows that 500 mM NaCl produces three peaks of autolyzed m-calpain, but in this instance, the center peak is the proteolytically active peak. The nature of the three m-calpain peaks is under investigation. Supported by the National Research Initiative Competitive Grants program, the Muscular Dystrophy Association, and the American Heart Association.

21)eat-1 Encodes Two Members of the ALP/Enigma Protein Family Required for Normal Muscle Function in Caenorhabditis elegans

Caroline R. McKeown, Mary C Beckerle, The Huntsman Cancer Institute and the Department of Biology, University of Utah, 2000 E. Circle of Hope, Salt Lake City, UT 84112

Muscle is required for many vital processes including locomotion, circulation and respiration.  Members of the alpha-actinin associated LIM protein (ALP)-Enigma family have been identified as prominent constituents of actin filament anchorage sites in vertebrate muscle and have been postulated to be involved in cardiomyopathy and facioscapulohumeral muscular dystrophy.  ALP-Enigma family members are comprised of N-terminal PDZ domains linked to one (ALP) or three (Enigma) C-terminal LIM domains.  Vertebrates express many closely related members of this protein family, making it difficult to analyze the roles of the individual proteins in vivo.  We have identified a single gene in C. elegans that, through alternative splicing, encodes both ALP and Enigma isoforms.  By generating transgenic animals that express GFP-tagged Enigma under the control of its endogenous promoter, we determined that C. elegans Enigma is expressed broadly in muscle derivatives, where it is concentrated at actin anchorage sites such as the dense bodies of body wall muscle.  Using both forward- and reverse-genetic approaches, we demonstrate that loss of ALP-Enigma in C. elegans results in widespread muscle defects that include problems with locomotion, pharyngeal pumping, and egg-laying.  Based on genetic mapping, RNA-interference, and genomic rescue data, we conclude that the ALP-Enigma proteins are encoded by the eat-1 locus.  Furthermore, we demonstrate that eat-1 mutants display defects in the muscle cytoskeleton that are likely due to defective actin anchorage.  Our findings illustrate that ALP-Enigma expression is absolutely required for normal muscle function in C. elegans.  With a single gene encoding all ALP-Enigma isoforms, C. elegans provides an excellent model for deciphering the functions of ALP and Enigma in vivo.

22) Coilin Forms the Bridge Between Cajal Bodies and SMN, the Spinal Muscular Atrophy Protein

A. Gregory Matera, Karen E. Tucker, Piotr W. Szymczyk, Karl B. Shpargel, Michael D. Hebert, Department of Genetics, Case Western Reserve Univ., 10900 Euclid Ave, Cleveland, OH 44106-4955

Spinal muscular atrophy (SMA) is a lethal genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1.  SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs).  In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin and Nopp140, the SMN protein complex, and the Cajal body (CB) signature protein, coilin.  Here we report on the generation of knockout mice lacking the C-terminal 487 aa of coilin and the role of this protein in intranuclear trafficking of the SMN complex.   We found that while, some homozygous mutant animals are viable, their numbers are significantly reduced when crossed to inbred backgrounds.  Analysis of tissues and cell lines from knockout animals revealed the presence of extranucleolar foci that contain fibrillarin and Nopp140 but not other typical nucleolar markers.  These so-called ‘residual’ CBs neither condense Sm proteins nor recruit members of the SMN protein complex.  Transient expression of wildtype mouse coilin in knockout cells resulted in formation of CBs and restored these missing epitopes.  Furthermore, we show that SMN interacts directly with coilin in vitro and in vivo, and that this interaction mediates recruitment of the SMN complex to CBs.  Mutation or deletion of specific arginine residues within coilin strongly inhibited the interaction.  Ectopic expression of SMN and coilin constructs in coilin knockout cells confirmed that recruitment of SMN and splicing snRNPs to CBs depends on a C-terminal RG dipeptide motif.  A cardinal cellular feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a pivotal role for coilin in this process.