TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.

AMPK Complex Activation Promotes Sarcolemmal Repair in Dysferlinopathy.

Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.

Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells.

Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast.

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.

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 Largemyd/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 (dysferlinsjl/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.

Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells.

Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro.

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

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.

Micromorphology of resin-dentin interfaces using one-bottle etch&rinse and self-etching adhesive systems on laser-treated dentin surfaces: a confocal laser scanning microscope analysis.

BACKGROUND AND OBJECTIVES: This study evaluated the hybrid layer (HL) morphology created by three adhesive systems (AS) on dentin surfaces treated with Er:YAG laser using two irradiation parameters. STUDY DESIGN: Occlusal flat dentin surfaces of 36 human third molars were assigned into nine groups (n = 4) according to the following ASs: one bottle etch&rinse Single Bond Plus (3M ESPE), two-step Clearfil Protect Bond (Kuraray), and all-in-one S(3) Bond (Kuraray) self-etching, which were labeled with rhodamine B or fluorescein isothiocyanate-dextran and were applied to dentin surfaces that were irradiated with Er:YAG laser at either 120 (38.7 J/cm(2)) or 200 mJ/pulse (64.5 J/cm(2)), or were applied to untreated dentin surfaces (control group). The ASs were light-activated following MI and the bonded surfaces were restored with resin composite Z250 (3M ESPE). After 24 hours of storage in vegetable oil, the restored teeth were vertically, serially sectioned into 1-mm thick slabs, which had the adhesive interfaces analyzed with confocal laser microscope (CLSM-LSM 510 Meta). CLSM images were recorded in the fluorescent mode from three different regions along each bonded interface. RESULTS: Non-uniform HL was created on laser-irradiated dentin surfaces regardless of laser irradiation protocol for all AS, while regular and uniform HL was observed in the control groups. "Stretch mark"-like red lines were found within the HL as a result of resin infiltration into dentin microfissures, which were predominantly observed in 200 mJ/pulse groups regardless of AS. Poor resin infiltration into peritubular dentin was observed in most regions of adhesive interfaces created by all ASs on laser-irradiated dentin, resulting in thin resin tags with neither funnel-shaped morphology nor lateral resin projections. CONCLUSION: Laser irradiation of dentin surfaces at 120 or 200 mJ/pulse resulted in morphological changes in HL and resin tags for all ASs evaluated in the study.

Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer.

Deficiency of the dysferlin protein presents as two major clinical phenotypes: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin is known to participate in membrane repair, providing a potential hypothesis to the underlying pathophysiology of these diseases. The size of the dysferlin cDNA prevents its direct incorporation into an adeno-associated virus (AAV) vector for therapeutic gene transfer into muscle. To bypass this limitation, we split the dysferlin cDNA at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Intramuscular injection of the corresponding vectors into a dysferlin-deficient mouse model led to the expression of full-length dysferlin for at least 1 year. Importantly, systemic injection in the tail vein of the two vectors led to a widespread although weak expression of the full-length protein. Injections were associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.

Calcium-dependent plasma membrane repair requires m- or mu-calpain, but not calpain-3, the proteasome, or caspases.

Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the non-catalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive E1 ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or mu-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury.

Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan.

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by alpha-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin alpha7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Large(myd) muscles, which have an intact dystrophin-glycoprotein complex and lack only the laminin globular domain-binding motif on alpha-dystroglycan. Compromised sarcolemmal integrity is directly shown in Large(myd) muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding alpha-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage.

Two-photon laser confocal microscopy of micropermeability of resin-dentin bonds made with water or ethanol wet bonding.

This study evaluated the micropermeability of six etch-and-rinse adhesives bonded to dentin. There were two principal groups: wet bonding with water or wet bonding with absolute ethyl alcohol. After bonding and the creation of composite build-ups, the pulp chambers were filled with 0.1% lucifer yellow. The contents of the pulp chamber were kept under 20 cm H(2)O pressure to simulate pulpal pressure for 3 h. The specimens were vertically sectioned into multiple 0.5-mm thick slabs that were polished and then examined using a two-photon confocal laser scanning microscope (TPCLSM). The results showed that specimens bonded with adhesives using the water wet-bonding condition all showed tracer taken up uniformly by the hybrid layer. This uptake of fluorescent tracer into the hybrid layer was quantified by computer software. The most hydrophobic experimental resins showed the highest fluorescent tracer uptake (ca. 1800 +/- 160 arbitrary fluorescent units/std. surface area). The most hydrophilic experimental resins showed the lowest tracer uptake into water-saturated hybrid layers. When ethanol wet-bonding was used, significantly less fluorescent tracer was seen in hybrid layers. The most hydrophilic experimental resins and Single Bond Plus showed little micropermeability. Clearly, ethanol wet-bonding seals dentin significantly better than water-wet dentin regardless of the adhesive in etch-and-rinse systems.

Lectin-based food poisoning: a new mechanism of protein toxicity.

BACKGROUND: Ingestion of the lectins present in certain improperly cooked vegetables can result in acute GI tract distress, but the mechanism of toxicity is unknown. In vivo, gut epithelial cells are constantly exposed to mechanical and other stresses and consequently individual cells frequently experience plasma membrane disruptions. Repair of these cell surface disruptions allows the wounded cell to survive: failure results in necrotic cell death. Plasma membrane repair is mediated, in part, by an exocytotic event that adds a patch of internal membrane to the defect site. Lectins are known to inhibit exocytosis. We therefore tested the novel hypothesis that lectin toxicity is due to an inhibitory effect on plasma membrane repair. METHODS AND FINDINGS: Repair of plasma membrane disruptions and exocytosis of mucus was assessed after treatment of cultured cell models and excised segments of the GI tract with lectins. Plasma membrane disruptions were produced by focal irradiation of individual cells, using a microscope-based laser, or by mechanical abrasion of multiple cells, using a syringe needle. Repair was then assessed by monitoring the cytosolic penetration of dyes incapable of crossing the intact plasma membrane. We found that cell surface-bound lectins potently inhibited plasma membrane repair, and the exocytosis of mucus that normally accompanies the repair response. CONCLUSIONS: Lectins potently inhibit plasma membrane repair, and hence are toxic to wounded cells. This represents a novel form of protein-based toxicity, one that, we propose, is the basis of plant lectin food poisoning.

Quantification of ionizing radiation-induced cell death in situ in a vertebrate embryo.

Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.

Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin.

Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling.

Sex steroids exert profound and controversial effects on cardiovascular function. For example, estrogens have been reported to either ameliorate or exacerbate coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, nongenomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMC). The purpose of this study was to elucidate nongenomic estrogen signal transduction in HCASMC. We have used tissue (arterial tension studies), cellular (single-channel patch clamp, fluorescence), and molecular (protein expression) techniques to now identify novel targets of estrogen action in HCASMC: type I (neuronal) nitric oxide synthase (nNOS) and phosphatidylinositol 3-kinase (PI3-kinase)Akt. 17beta-Estradiol (E(2)) increased NO-stimulated fluorescence in HCASMC, and cell-attached patch-clamp experiments revealed that stimulation of nNOS leads to increased activity of calcium-activated potassium (BK(Ca)) channels in these cells. Furthermore, overexpression of nNOS protein in HCASMC greatly enhanced BK(Ca) channel activity. Immunoblot studies demonstrated that E(2) enhances Akt phosphorylation in HCASMC and that wortmannin, an inhibitor of PI3-kinase, attenuated E(2)-stimulated channel activity, NO production, Akt phosphorylation, and estrogen-stimulated coronary relaxation. These studies implicate the PI3-kinase/Akt signaling axis as an estrogen transduction component in vascular smooth muscle cells. We conclude, therefore, that estrogen opens BK(Ca) channels in HCASMC by stimulating nNOS via a transduction sequence involving PI3-kinase and Akt. These findings now provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.

Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.

Two autosomal recessive muscle diseases, limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are caused by mutations in the dysferlin gene. These mutations result in poor ability to repair cell membrane damage, which is suggested to be the cause for this disease. However, many patients who share clinical features with MM-type muscular dystrophy do not carry mutations in dysferlin gene. To understand the basis of MM that is not due to mutations in dysferlin gene, we analyzed cells from patients in one such family. In these patients, we found no defects in several potential candidates - annexin A2, caveolin-3, myoferlin and the MMD2 locus on chromosome 10p. Similar to dysferlinopathy, these cells also exhibit membrane repair defects and the severity of the defect correlated with severity of their disease. However, unlike dysferlinopathy, none of the conventional membrane repair pathways are defective in these patient cells. These results add to the existing evidence that cell membrane repair defect may be responsible for MM-type muscular dystrophy and indicate that a previously unsuspected genetic lesion that affects cell membrane repair pathway is responsible for the disease in the non-dysferlin MM patients.

Calpain is required for the rapid, calcium-dependent repair of wounded plasma membrane.

Mammalian cells require extracellular calcium ion to undergo rapid plasma membrane repair seconds after mechanical damage. Utilizing transformed fibroblasts from calpain small subunit knock-out (Capns1-/-) mouse embryos, we now show that the heterodimeric, typical subclass of calpains is required for calcium-mediated survival after plasma membrane damage caused by scraping a cell monolayer. Survival of scrape-damaged Capns1-/- cells was unaffected by calcium in the scraping medium, whereas more Capns1+/+ cells survived when calcium was present. Calcium-mediated survival was increased when Capns1-/- cells were scraped in the presence of purified m- or mu-calpain. Survival rates of scraped Capns1+/+, HFL-1, or Chinese hamster ovary cells were decreased by the calpain inhibitor, calpeptin, or the highly specific calpain inhibitor protein, calpastatin. Capns1-/- cells failed to reseal following laser-induced membrane disruption, demonstrating that their decreased survival after scraping resulted, at least in part, from failed membrane repair. Proteomic and immunologic analyses demonstrated that the known calpain substrates talin and vimentin were exposed at the cell surface and processed by calpain following cell scraping. Autoproteolytic activation of calpain at the scrape site was evident at the earliest time point analyzed and appeared to precede proteolysis of talin and vimentin. The results indicate that conventional calpains are required for calcium-facilitated survival after plasma membrane damage and may act by localized remodeling of the cortical cytoskeleton at the injury site.

Conditional ablation of MHC-II suggests an indirect role for MHC-II in regulatory CD4 T cell maintenance.

Although the importance of MHC class II (MHC-II) in acute homeostatic proliferation of regulatory T (Treg) cells has been established, we considered here the maintenance and state of Treg cells in mice that are almost completely devoid of MHC-II in their periphery but still make their own CD4 T cells and Treg cells. The latter was accomplished by conditional deletion of a loxP-flanked MHC-II beta-chain allele using a TIE2Cre transgene, which causes a very high degree of deletion in hemopoietic/endothelial progenitor cells but without deletion among thymic epithelial cells. Such conditional MHC-II-deficient mice possess their own relatively stable levels of CD4+CD25+ cells, with a normal fraction of Foxp3+ Treg cells therein, but at a level approximately 2-fold lower than in control mice. Thus, both Foxp3low/- CD4+CD25+ cells, said to be a major source of IL-2, and IL-2-dependent Foxp3+ Treg cells are reduced in number. Furthermore, CD25 expression is marginally reduced among Foxp3+ Treg cells in conditional MHC-II-deficient mice, indicative of a lack of MHC-II-dependent TCR stimulation and/or IL-2 availability, and IL-2 administration in vivo caused greatly increased cell division among adoptively transferred Treg cells. This is not to say that IL-2 can cause Treg cell division in the complete absence of MHC-II as small numbers of MHC-II-bearing cells do remain in conditional MHC-II-deficient mice. Rather, this suggests only that IL-2 was limiting. Thus, our findings lend support to the proposal that Treg cell homeostasis depends on a delicate balance with a population of self-reactive IL-2-producing CD4+CD25+ cells which are themselves at least in part MHC-II-dependent.