The Role of ß-Dystroglycan in Nuclear Dynamics.

Dystroglycan is a ubiquitously expressed heterodimeric cell-surface laminin receptor with roles in cell adhesion, signalling, and membrane stabilisation. More recently, the transmembrane ß-subunit of dystroglycan has been shown to localise to both the nuclear envelope and the nucleoplasm. This has led to the hypothesis that dystroglycan may have a structural role at the nuclear envelope analogous to its role at the plasma membrane. The biochemical fraction of myoblast cells clearly supports the presence of dystroglycan in the nucleus. Deletion of the dystroglycan protein by disruption of the DAG1 locus using CRISPR/Cas9 leads to changes in nuclear size but not overall morphology; moreover, the Young's modulus of dystroglycan-deleted nuclei, as determined by atomic force microscopy, is unaltered. Dystroglycan-disrupted myoblasts are also no more susceptible to nuclear stresses including chemical and mechanical, than normal myoblasts. Re-expression of dystroglycan in DAG1-disrupted myoblasts restores nuclear size without affecting other nuclear parameters.

The Molecular Basis and Biologic Significance of the ß-Dystroglycan-Emerin Interaction.

ß-dystroglycan (ß-DG) assembles with lamins A/C and B1 and emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of ß-DG, we characterized the interaction between ß-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery-Dreifuss muscular dystrophy (EDMD). Using truncated variants of ß-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the ß-DG-emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to ß-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of ß-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. ß-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that ß-DG plays a role as an emerin interacting partner modulating its stability and function.

How the central domain of dystrophin acts to bridge F-actin to sarcolemmal lipids.

Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin/dystrophin/membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein.

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity.

The study of Hutchinson-Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin-driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1-target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.

The intracellular domain of ß-dystroglycan mediates the nucleolar stress response by suppressing UBF transcriptional activity.

ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.

Publisher Correction: Retrograde trafficking of ß-dystroglycan from the plasma membrane to the nucleus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Are Soy Products Effective in DMD?

INTRODUCTION: In addition to their nutritional value, processed soy bean extracts contain several activities with potential therapeutic benefits. These include anti-oxidants, and tyrosine kinase and protease inhibitory activity. There are also anecdotal reports of health benefits of soy products in alleviating DMD symptoms. METHODS: Mdx mice were fed a control soy-free diet or the same diet containing either a proprietary soy preparation (Haelan 951), purified soy isoflavones, purified Bowman-Birk protease inhibitor or a combination of isoflavones and Bowman-Birk inhibitor. Mice were tested for their wire hanging ability at the start of the diet regimen and every 4 weeks until week 12 of treatment. RESULTS AND DISCUSSION: The diet containing Bowman-Birk inhibitor was the only one to show a significant and sustained improvement over the 12 weeks of the study. All other dietary additions; Haelan 951, isoflavones and isoflavones with Bowman-Birk inhibitor, were not significantly different from each other or from control. The effectiveness of Bowman-Birk inhibitor in mdx mice clearly warrants further study.

Control of nuclear ß-dystroglycan content is crucial for the maintenance of nuclear envelope integrity and function.

ß-Dystroglycan (ß-DG) is a plasma membrane protein that has ability to target to the nuclear envelope (NE) to maintain nuclear architecture. Nevertheless, mechanisms controlling ß-DG nuclear localization and the physiological consequences of a failure of trafficking are largely unknown. We show that ß-DG has a nuclear export pathway in myoblasts that depends on the recognition of a nuclear export signal located in its transmembrane domain, by CRM1. Remarkably, NES mutations forced ß-DG nuclear accumulation resulting in mislocalization and decreased levels of emerin and lamin B1 and disruption of various nuclear processes in which emerin (centrosome-nucleus linkage and ß-catenin transcriptional activity) and lamin B1 (cell cycle progression and nucleoli structure) are critically involved. In addition to nuclear export, the lifespan of nuclear ß-DG is restricted by its nuclear proteasomal degradation. Collectively our data show that control of nuclear ß-DG content by the combination of CRM1 nuclear export and nuclear proteasome pathways is physiologically relevant to preserve proper NE structure and activity.

Retrograde trafficking of ß-dystroglycan from the plasma membrane to the nucleus.

ß-Dystroglycan (ß-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of ß-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/ß mediated nuclear import pathway has already been described for ß-DG, the intracellular trafficking route by which ß-DG reaches the nucleus is unknown. In this study, we demonstrated that ß-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of ß-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of ß-DG at Tyr890 is a key stimulus for ß-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that ß-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.

A role for dystroglycan in the pathophysiology of acute leukemic cells.

AIMS: Previous reports have demonstrated that alterations or reduced expression of Dystroglycan (Dg) complex (αDg and ßDg subunits) are related to progression and severity of neoplastic solid tissues. Therefore we determined the expression pattern and subcellular distribution of Dg complex in Acute Myeloid Leukemia (AML) primary blasts (M1, M2, and M3 phenotypes), as well as HL-60 and Kasumi-1 leukemia cell lines. Additionally, we evaluated the relative expression of the main enzymes controlling α-Dg glycosylation to ascertain the post-translational modifications in the leukemia cell phenotype. MAIN METHODS: Primary leukemia blasts and leukemia cell lines were processed by confocal analysis to determine the subcellular distribution of α-Dg, ß-Dg, and phosphorylated ß-Dg (Y892), to evaluate the expression pattern of the different Dg species we performed Western Blot (WB) assays, while the messenger RNA (mRNA) expression of enzymes involved in α-Dg glycosylation, such as POMGnT1, POMT1, POMT2, LARGE, FKTN, and FKRP, were evaluated by qualitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, in an attempt to ameliorate the leukemia cell phenotype, we transfected leukemia cells with a plasmid expressing the Dg complex. KEY FINDINGS: The Dg complex was altered in leukemia cells, including decreased mRNA, protein, and α-Dg glycosylated levels, mislocalization of ß-Dg, and a diminution of mRNA expression of LARGE in patients leukemia blasts and in cell lines. Interestingly, the exogenous expression of Dg complex promoted filopodial formation, differentiation, and diminished proliferation, attenuating some HL-60 and Kasumi cells characteristics. SIGNIFICANCE: Dg complex integrity and balance are required for a proper hematopoietic cell function, in that its disruption might contribute to leukemia pathophysiology.

γ-Secretase Dependent Nuclear Targeting of Dystroglycan.

Dystroglycan is frequently lost in adenocarcinoma. α-dystroglycan is known to become hypoglycosylated due to transcriptional silencing of LARGE, whereas ß-dystroglycan is proteolytically cleaved and degraded. The mechanism and proteases involved in the cleavage events affecting ß-dystroglycan are poorly understood. Using LNCaP prostate cancer cells as a model system, we have investigated proteases and tyrosine phosphorylation affecting ß-dystroglycan proteolysis and nuclear targeting. Cell density or phorbol ester treatment increases dystroglycan proteolysis, whereas furin or γ-secretase inhibitors decreased dystroglycan proteolysis. Using resveratrol treatment of LNCaP cells cultured at low cell density in order to up-regulate notch and activate proteolysis, we identified significant increases in the levels of a 26 kDa ß-dystroglycan fragment. These data, therefore, support a cell density-dependent γ-secretase and furin mediated proteolysis of ß-dystroglycan, which could be notch stimulated, leading to nuclear targeting and subsequent degradation. 117: 2149-2157, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

The synthesis and biological evaluation of a kabiramide C fragment modified with a WH2 consensus actin-binding motif as a potential disruptor of the actin cytoskeleton.

The F-actin depolymerisation potency of a fragment of kabiramide C was increased when modified with a WH2 consensus actin-binding motif LKKV. Despite its low affinity for actin monomers, a shorter analogous fragment not bearing LKKV was identified as a potent inhibitor of actin polymerisation and a promoter of its depolymerisation, resulting in a loss of actin stress fibres in cells.

Dasatinib as a treatment for Duchenne muscular dystrophy.

Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of ß-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of ß-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic sapje zebrafish, we have investigated the use of tyrosine kinase and other inhibitors to treat the dystrophic symptoms in this model of Duchenne muscular dystrophy. Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of ß-dystroglycan phosphorylation on tyrosine and to increase the relative levels of non-phosphorylated ß-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of ß-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy.

A Modified Wire Hanging Apparatus for Small Animal Muscle Function Testing.

Wire hang tests are simple and cheap methods to assess muscle performance in small rodents, but do not always yield consistent results. We describe a simple wire hang apparatus that comprises a continuous rolling loop. Wire hang times measured using the rolling wire provide consistent and reliable data that more accurately reflect the output of a continuous physical effort. As such data obtained in mice using a rolling wire are more representative of the physical changes in the mouse muscle and less susceptible to individual mouse behaviour and differences in animal handling.

Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells.

BACKGROUND: Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils. PURPOSE: In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells. METHODS: We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells. RESULTS: Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1ß and expression of markers of differentiation are not altered. CONCLUSION: Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.

Laminin promotes metalloproteinase-mediated dystroglycan processing to regulate oligodendrocyte progenitor cell proliferation.

The cell surface receptor dystroglycan mediates interactions between oligodendroglia and laminin-211, an extracellular matrix protein that regulates timely oligodendroglial development. However, dystroglycan's precise role in oligodendroglial development and the potential mechanisms to regulate laminin-dystroglycan interactions remain unknown. Here we report that oligodendroglial dystroglycan is cleaved by metalloproteinases, thereby uncoupling oligodendroglia from laminin binding. Dystroglycan cleavage is selectively stimulated by oligodendrocyte progenitor cell attachment to laminin-211, but not laminin-111 or poly-D-lysine. In addition, dystroglycan cleavage occurs most prominently in oligodendrocyte progenitor cells, with limited dystroglycan cleavage observed in differentiating oligodendrocytes. When dystroglycan cleavage is blocked by metalloproteinase inhibitors, oligodendrocyte progenitor cell proliferation is substantially decreased. Conversely, expression of the intracellular portion of cleaved dystroglycan results in increased oligodendrocyte progenitor cell proliferation, suggesting that endogenous dystroglycan cleavage may promote oligodendrocyte progenitor cell cycle progression. Intriguingly, while matrix metalloproteinase-2 and/or -9 have been reported to be responsible for dystroglycan cleavage, we find that these two metalloproteinases are neither necessary nor sufficient for cleavage of oligodendroglial dystroglycan. In summary, laminin-211 stimulates metalloproteinase-mediated dystroglycan cleavage in oligodendrocyte progenitor cells (but not in differentiated oligodendrocytes), which in turn promotes oligodendrocyte progenitor cell proliferation. This novel regulation of oligodendroglial laminin-dystroglycan interactions may have important consequences for oligodendroglial differentiation, both during development and during disease when metalloproteinase levels become elevated.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and ß-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete ß-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for ß-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

The actin binding affinity of the utrophin tandem calponin-homology domain is primarily determined by its N-terminal domain.

The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.