Ivette Martínez-Vieyra

A role for β-dystroglycan in the organization and structure of the nucleus in myoblasts

We recently characterized a nuclear import pathway for β-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of β-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that β-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of β-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that β-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of β-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that β-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, β-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that β-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies.

Characterization of an Importin α/β-recognized nuclear localization signal in β-dystroglycan†

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R779 and K780 are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y892 playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010.

Epithelial sodium channel modulates platelet collagen activation

Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation.

Distribution of dystrophin‐ and utrophin‐associated protein complexes (DAPC/UAPC) in human hematopoietic stem/progenitor cells

Hematopoietic stem cells (HSC) are defined by their cardinal properties, such as sustained proliferation, multilineage differentiation, and self‐renewal, which give rise to a hierarchy of progenitor populations with more restricted potential lineage, ultimately leading to the production of all types of mature blood cells. HSC are anchored by cell adhesion molecules to their specific microenvironment, thus regulating their cell cycle, while cell migration is essentially required for seeding the HSC of the fetal bone marrow (BM) during development as well as in adult BM homeostasis. The dystrophin‐associated protein complex (DAPC) is a large group of membrane‐associated proteins linking the cytoskeleton to the extracellular matrix and exhibiting scaffolding, adhesion, and signaling roles in muscle and non‐muscle cells including mature blood cells. Because adhesion and migration are mechanisms that influence the fate of the HSC, we explored the presence and the feasible role of DAPC. In this study, we characterized the pattern expression by immunoblot technique and, by confocal microscopy analysis, the cellular distribution of dystrophin and utrophin gene products, and the dystrophin‐associated proteins (α‐, β‐dystroglycan, α‐syntrophin, α‐dystrobrevin) in relation to actin filaments in freshly isolated CD34+ cells from umbilical cord blood. Immunoprecipitation assays demonstrated the presence of Dp71d/Dp71Δ110m∼DAPC and Up400/Up140∼DAPC. The subcellular distribution of the two DAPC in actin‐based structures suggests their dynamic participation in adhesion and cell migration. In addition, the particular protein pattern expression found in hematopoietic stem/progenitor cells might be indicative of their feasible participation during differentiation.

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.

Haemostatic role of intermediate filaments in adhered platelets: importance of the membranous system stability.

The role of platelets in coagulation and the haemostatic process was initially suggested two centuries ago, and under appropriate physiological stimuli, these undergo abrupt morphological changes, attaching and spreading on damaged endothelium, preventing bleeding. During the adhesion process, platelet cytoskeleton reorganizes generating compartments in which actin filaments, microtubules, and associated proteins are arranged in characteristic patterns mediating crucial events, such as centralization of their organelles, secretion of granule contents, aggregation with one another to form a haemostatic plug, and retraction of these aggregates. However, the role of Intermediate filaments during the platelet adhesion process has not been explored. J. Cell. Biochem. 114: 2050–2060, 2013.

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-1β 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.

Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

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 β‐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 β‐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 β‐dystroglycan. In conclusion, caveolin‐1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. J. Cell. Biochem. 116: 2528–2540, 2015.

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 (&agr;Dg and &bgr;Dg 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 &agr;‐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 &agr;‐Dg, &bgr;‐Dg, and phosphorylated &bgr;‐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 &agr;‐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 &agr;‐Dg glycosylated levels, mislocalization of &bgr;‐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. Graphical abstract Figure. No caption available.