Variation in mutation, recombination, and transposition rates in Drosophila melanogaster and Drosophila simulans.

The rates of mutation, recombination, and transposition are core parameters in models of evolution. They impact genetic diversity, responses to ongoing selection, and levels of genetic load. However, even for key evolutionary model species such as Drosophila melanogaster and Drosophila simulans, few estimates of these parameters are available, and we have little idea of how rates vary between individuals, sexes, or populations. Knowledge of this variation is fundamental for parameterizing models of genome evolution. Here, we provide direct estimates of mutation, recombination, and transposition rates and their variation in a West African and a European population of D. melanogaster and a European population of D. simulans Across 89 flies, we observe 58 single-nucleotide mutations, 286 crossovers, and 89 transposable element (TE) insertions. Compared to the European D. melanogaster, we find the West African population has a lower mutation rate (1.67 × 10-9 site-1 gen-1 vs. 4.86 × 10-9 site-1 gen-1) and a lower transposition rate (8.99 × 10-5 copy-1 gen-1 vs. 23.36 × 10-5 copy-1 gen-1), but a higher recombination rate (3.44 cM/Mb vs. 2.06 cM/Mb). The European D. simulans population has a similar mutation rate to European D. melanogaster, but a significantly higher recombination rate and a lower, but not significantly different, transposition rate. Overall, we find paternal-derived mutations are more frequent than maternal ones in both species. Our study quantifies the variation in rates of mutation, recombination, and transposition among different populations and sexes, and our direct estimates of these parameters in D. melanogaster and D. simulans will benefit future studies in population and evolutionary genetics.

Decreased pericellular matrix production and selection for enhanced cell membrane repair may impair osteocyte responses to mechanical loading in the aging skeleton.

Transient plasma membrane disruptions (PMD) occur in osteocytes with in vitro and in vivo loading, initiating mechanotransduction. The goal here was to determine whether osteocyte PMD formation or repair is affected by aging. Osteocytes from old (24 months) mice developed fewer PMD (-76% females, -54% males) from fluid shear than young (3 months) mice, and old mice developed fewer osteocyte PMD (-51%) during treadmill running. This was due at least in part to decreased pericellular matrix production, as studies revealed that pericellular matrix is integral to formation of osteocyte PMD, and aged osteocytes produced less pericellular matrix (-55%). Surprisingly, osteocyte PMD repair rate was faster (+25% females, +26% males) in osteocytes from old mice, and calcium wave propagation to adjacent nonwounded osteocytes was blunted, consistent with impaired mechanotransduction downstream of PMD in osteocytes with fast PMD repair in previous studies. Inducing PMD via fluid flow in young osteocytes in the presence of oxidative stress decreased postwounding cell survival and promoted accelerated PMD repair in surviving cells, suggesting selective loss of slower-repairing osteocytes. Therefore, as oxidative stress increases during aging, slower-repairing osteocytes may be unable to successfully repair PMD, leading to slower-repairing osteocyte death in favor of faster-repairing osteocyte survival. Since PMD are an important initiator of mechanotransduction, age-related decreases in pericellular matrix and loss of slower-repairing osteocytes may impair the ability of bone to properly respond to mechanical loading with bone formation. These data suggest that PMD formation and repair mechanisms represent new targets for improving bone mechanosensitivity with aging.

Inhibition of Osteocyte Membrane Repair Activity via Dietary Vitamin E Deprivation Impairs Osteocyte Survival.

Osteocytes experience plasma membrane disruptions (PMD) that initiate mechanotransduction both in vitro and in vivo in response to mechanical loading, suggesting that osteocytes use PMD to sense and adapt to mechanical stimuli. PMD repair is crucial for cell survival; antioxidants (e.g., alpha-tocopherol, also known as Vitamin E) promote repair while reactive oxygen species (ROS), which can accumulate during exercise, inhibit repair. The goal of this study was to determine whether depleting Vitamin E in the diet would impact osteocyte survival and bone adaptation with loading. Male CD-1 mice (3 weeks old) were fed either a regular diet (RD) or Vitamin E-deficient diet (VEDD) for up to 11 weeks. Mice from each dietary group either served as sedentary controls with normal cage activity, or were subjected to treadmill exercise (one bout of exercise or daily exercise for 5 weeks). VEDD-fed mice showed more PMD-affected osteocytes (+ 50%) after a single exercise bout suggesting impaired PMD repair following Vitamin E deprivation. After 5 weeks of daily exercise, VEDD mice failed to show an exercise-induced increase in osteocyte PMD formation, and showed signs of increased osteocytic oxidative stress and impaired osteocyte survival. Surprisingly, exercise-induced increases in cortical bone formation rate were only significant for VEDD-fed mice. This result may be consistent with previous studies in skeletal muscle, where myocyte PMD repair failure (e.g., with muscular dystrophy) initially triggers hypertrophy but later leads to widespread degeneration. In vitro, mechanically wounded MLO-Y4 cells displayed increased post-wounding necrosis (+ 40-fold) in the presence of H2O2, which could be prevented by Vitamin E pre-treatment. Taken together, our data support the idea that antioxidant-influenced osteocyte membrane repair is a vital aspect of bone mechanosensation in the osteocytic control of PMD-driven bone adaptation.

Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone.

Osteocytes sense loading in bone, but their mechanosensation mechanisms remain poorly understood. Plasma membrane disruptions (PMD) develop with loading under physiological conditions in many cell types (e.g., myocytes, endothelial cells). These PMD foster molecular flux across cell membranes that promotes tissue adaptation, but this mechanosensation mechanism had not been explored in osteocytes. Our goal was to investigate whether PMD occur and initiate consequent mechanotransduction in osteocytes during physiological loading. We found that osteocytes experience PMD during in vitro (fluid flow) and in vivo (treadmill exercise) mechanical loading, in proportion to the level of stress experienced. In fluid flow studies, osteocyte PMD preferentially formed with rapid as compared to gradual application of loading. In treadmill studies, osteocyte PMD increased with loading in weight bearing locations (tibia), but this trend was not seen in non-weight bearing locations (skull). PMD initiated osteocyte mechanotransduction including calcium signaling and expression of c-fos, and repair rates of these PMD could be enhanced or inhibited pharmacologically to alter downstream mechanotransduction and osteocyte survival. PMD may represent a novel mechanosensation pathway in bone and a target for modifying skeletal adaptation signaling in osteocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:653-662, 2018.

Environmental concentrations of prednisolone alter visually mediated responses during early life stages of zebrafish (Danio rerio).

The development of the eye in vertebrates is dependent upon glucocorticoid signalling, however, specific components of the eye are sensitive to synthetic glucocorticoids. The presence of synthetic glucocorticoids within the aquatic environment may therefore have important consequences for fish, which are heavily reliant upon vision for mediating several key behaviours. The potential ethological impact of synthetic glucocorticoid oculotoxicity however has yet to be studied. Physiological and behavioural responses which are dependent upon vision were selected to investigate the possible toxicity of prednisolone, a commonly occurring synthetic glucocorticoid within the environment, during early life stages of zebrafish. Although exposure to prednisolone did not alter the morphology of the external eye, aggregation of melanin within the skin in response to increasing light levels was impeded and embryos exposed to prednisolone (10 µg/l) maintained a darkened phenotype. Exposure to prednisolone also increased the preference of embryos for a dark environment within a light dark box test in a concentration dependent manner. However the ability of embryos to detect motion appeared unaffected by prednisolone. Therefore, while significant effects were detected in several processes mediated by vision, changes occurred in a manner which suggest that vision was in itself unaffected by prednisolone. Neurological and endocrinological changes during early ontogeny are considered as likely candidates for future investigation.

Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

Physiological and Behavioral Effects of Exposure to Environmentally Relevant Concentrations of Prednisolone During Zebrafish (Danio rerio) Embryogenesis.

The presence of synthetic glucocorticoids within the aquatic environment has been highlighted as a potential environmental concern as they may mimic the role of endogenous glucocorticoids during vertebrate ontogeny. Prednisolone is a commonly prescribed synthetic glucocorticoid which has been repeatedly detected in the environment. This study investigated the impact of environmentally relevant concentrations of prednisolone (0.1, 1, and 10 µg/L) during zebrafish embryogenesis using physiological and behavioral end points which are known to be mediated by endogenous glucocorticoids. The frequency of spontaneous muscle contractions (24 hpf) was significantly reduced by prednisolone and 0.1 µg/L increased the distance embryos swam in response to a mechanosensory stimulus (48 hpf). The percentage of embryos hatched significantly increased following prednisolone treatment (1 and 10 µg/L), while growth and mortality were unaffected. The onset of heart contraction was differentially affected by prednisolone while heart rate and oxygen consumption both increased significantly throughout embryogenesis. No substantial effect on the axial musculature was observed. Morphological changes to the lower jaw were detected at 96 hpf in response to 1 µg/L of prednisolone. Several parameters of swim behavior were also significantly affected. Environmentally relevant concentrations of prednisolone therefore alter early zebrafish ontogeny and significantly affect embryo behavior.

Membrane Repair: Mechanisms and Pathophysiology.

Eukaryotic cells have been confronted throughout their evolution with potentially lethal plasma membrane injuries, including those caused by osmotic stress, by infection from bacterial toxins and parasites, and by mechanical and ischemic stress. The wounded cell can survive if a rapid repair response is mounted that restores boundary integrity. Calcium has been identified as the key trigger to activate an effective membrane repair response that utilizes exocytosis and endocytosis to repair a membrane tear, or remove a membrane pore. We here review what is known about the cellular and molecular mechanisms of membrane repair, with particular emphasis on the relevance of repair as it relates to disease pathologies. Collective evidence reveals membrane repair employs primitive yet robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily honed for simplicity and success. Yet to be fully understood is whether core membrane repair machinery exists in all cells, or whether evolutionary adaptation has resulted in multiple compensatory repair pathways that specialize in different tissues and cells within our body.

The antioxidant requirement for plasma membrane repair in skeletal muscle.

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of the pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.

Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish (Danio rerio).

The unique physicochemistry and potential toxicity of manufactured nanoparticles (NPs) requires innovative approaches for the assessment of toxicity to aquatic organisms. Here, the toxicity of Cu-NPs, Ag-NPs and TiO2-NPs on the lateral line system of free-swimming zebrafish embryos was investigated and compared to appropriate metal salts or bulk material controls. Fish were exposed for 4h at 96-h post-fertilization. Metal salt (CuSO4 and AgNO3) controls reduced the number of functional lateral line neuromasts (LLN) to <5% of unexposed controls, but no effect on LLN was observed for TiO2-NPs or Ag-NPs. Exposure to Cu-NPs caused only a 15% reduction in LLN. Performance of positive rheotaxis was reduced by Cu-NPs, Ag-NPs, and the metal salt controls. The data show that some metal NPs can affect LLN and fish behaviour (rheotaxis) important for survival, and that effects were different from those of comparable metal ion controls. Capsule: We demonstrate that behaviour is a particularly sensitive indicator of metal NP exposure in fish and highlight the interaction between behaviour and external tissue surfaces.

Cell biology: ESCRTing trouble out!

Calcium entry through a plasma membrane defect leads to the local recruitment of endosomal complex required for transport (ESCRT) proteins. These proteins are hypothesized to drive an outward bending of the affected plasma membrane, forming a small bud that is then shed from the cell, along with the troublesome defect.

Cell wounding activates phospholipase D in primary mouse keratinocytes.

Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing.

Helicobacter pylori disrupts host cell membranes, initiating a repair response and cell proliferation.

Helicobacter pylori (H. pylori), the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA) have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+), single mutant (ΔvacA or ΔcagA) or double mutant (ΔvacA/ΔcagA) strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca(2+) influx. Ca(2+)-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

Promotion of plasma membrane repair by vitamin E.

Severe vitamin E deficiency results in lethal myopathy in animal models. Membrane repair is an important myocyte response to plasma membrane disruption injury as when repair fails, myocytes die and muscular dystrophy ensues. Here we show that supplementation of cultured cells with α-tocopherol, the most common form of vitamin E, promotes plasma membrane repair. Conversely, in the absence of α-tocopherol supplementation, exposure of cultured cells to an oxidant challenge strikingly inhibits repair. Comparative measurements reveal that, to promote repair, an anti-oxidant must associate with membranes, as α-tocopherol does, or be capable of α-tocopherol regeneration. Finally, we show that myocytes in intact muscle cannot repair membranes when exposed to an oxidant challenge, but show enhanced repair when supplemented with vitamin E. Our work suggests a novel biological function for vitamin E in promoting myocyte plasma membrane repair. We propose that this function is essential for maintenance of skeletal muscle homeostasis.

A novel cellular defect in diabetes: membrane repair failure.

OBJECTIVE: Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes. RESEARCH DESIGN AND METHODS: Myocytes in intact muscle from type 1 (INS2(Akita+/-)) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect. RESULTS: Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE. CONCLUSIONS: Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

Receptor for advanced glycation end products (RAGE) prevents endothelial cell membrane resealing and regulates F-actin remodeling in a beta-catenin-dependent manner.

Receptor for advanced glycation end products (RAGE), an immunoglobin superfamily cell surface receptor, contributes to the vascular pathology associated with multiple disorders, including Alzheimer disease (AD), diabetic complications, and inflammatory conditions. However, the underlying mechanisms remain largely unclear. Here, using the human umbilical vein endothelial cell line (ECV-304) expressing human RAGE, we report that RAGE expression leads to an altered F-actin organization and impaired membrane resealing. To investigate the underlying mechanisms, we showed that RAGE expression increases ß-catenin level, which decreases F-actin stress fibers and attenuates plasma membrane resealing. These results thus suggest a negative function for RAGE in endothelial cell membrane repair and reveal a new mechanism underlying RAGE regulation of F-actin remodeling and membrane resealing.

The role of N-methyl-D-aspartate receptor activation in homocysteine-induced death of retinal ganglion cells.

PURPOSE: Elevated plasma homocysteine has been implicated in glaucoma, a vision disorder characterized by retinal ganglion cell death. The toxic potential of homocysteine to ganglion cells is known, but the mechanisms are not clear. A mechanism of homocysteine-induced death of cerebral neurons is via N-methyl-D-aspartate (NMDA) receptor overstimulation, leading to excess calcium influx and oxidative stress. This study examined the role of the NMDA receptor in homocysteine-mediated ganglion cell death. METHODS: Primary mouse ganglion cells were used for these experiments. NMDA receptor stimulation by homocysteine was determined by patch clamp analysis and fluorescent detection of intracellular calcium. NMDA receptor involvement in homocysteine-mediated cell death was determined through assessment of lactate dehydrogenase release and TUNEL analysis. These experiments used the NMDA receptor blocker MK-801. Induction of reactive species superoxide, nitric oxide, and peroxynitrite was measured by electron paramagnetic resonance spectroscopy, chemiluminescent nitric oxide detection, and immunoblotting for nitrotyrosine, respectively. RESULTS: 50 µM homocysteine stimulated the NMDA receptor in presence of 100 µM glycine. Homocysteine induced 59.67 ± 4.89% ganglion cell death that was reduced to 19.87 ± 3.03% with cotreatment of 250 nM MK-801. Homocysteine elevated intracellular calcium ∼7-fold, which was completely prevented by MK-801. Homocysteine treatment increased superoxide and nitric oxide levels by ∼40% and ∼90%, respectively, after 6 hours. Homocysteine treatment elevated peroxynitrite by ∼85% after 9 hours. CONCLUSIONS: These experiments provide compelling evidence that homocysteine induces retinal ganglion cell toxicity through direct NMDA receptor stimulation and implicate, for the first time, the induction of oxidative stress as a potent mechanism of homocysteine-mediated ganglion cell death.

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.