Doris Cerecedo

Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

Role of dystrophins and utrophins in platelet adhesion process

Platelets are crucial at the site of vascular injury, adhering to the sub‐endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro‐coagulant factors contained in the granules and to constrict the clot. The interaction of F‐actin with different actin‐binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin‐associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin β‐1 fraction and the focal adhesion system (α‐actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin‐associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin‐associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

β‐Dystroglycan modulates the interplay between actin and microtubules in human‐adhered platelets

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full‐spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co‐ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin‐associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross‐talk between these two cytoskeletal systems. Interestingly, β‐dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co‐participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

Distribution of dystrophin- and utrophin-associated protein complexes during activation of human neutrophils

OBJECTIVE Dystrophins, utrophins, and their associated proteins are involved in structural and signaling roles in nonmuscle tissues; however, description of these proteins in neutrophils remained unexplored. Therefore we characterize the pattern expression, and the cellular distribution of dystrophin and utrophin gene products and dystrophin-associated proteins (i.e., beta-dystroglycan, alpha-syntrophin, and alpha-dystrobrevins) in relation to actin filaments in resting and activated with formyl-methionyl-leucyl-phenylalanine human neutrophils. MATERIALS AND METHODS Resting and fMLP-activated human neutrophils were analyzed by immunoblot and by confocal microscopy analysis. Immunoprecipitation assays were performed to corroborate the presence of protein complexes. RESULTS Immunoprecipitation assays and confocal analysis demonstrated the presence of two dystrophin-associated protein complexes in resting and activated neutrophils: the former formed by Dp71d/Dp71Delta(110)(m) and dystrophin-associated proteins (beta-dystroglycan, alpha-syntrophin, alpha-dystrobrevin-1, and -2), while the latter contains Up400, instead of Dp71d/Dp71Delta(110)(m), as a central component of the dystrophin-associated protein complexes (DAPC). Confocal analysis also showed the subcellular redistribution of Dp71d/Dp71Delta(110)(m) approximately DAPC and Up400 approximately DAPC in F-actin-based structures displayed during activation process with fMLP. CONCLUSIONS Our study showed the existence of two protein complexes formed by Dp71d/Dp71Delta(110)(m) or Up400 associated with DAPs in resting and fMLP-treated human polymorphonuclears. The interaction of these complexes with the actin cytoskeleton is indicative of their dynamic participation in the chemotaxis process.

Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of α-dystrobrevins

Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension‐activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. α‐ and β‐dystrobrevin members of the dystrophin‐associated protein complex in muscle and non‐muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton‐disrupting drugs, quantification of soluble P‐selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of α‐dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which α‐dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

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.

Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

Platelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx3cv platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Δ110m and dystrophin-associated protein in adhered platelets from dystrophic mdx3cv mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx3cv platelets.

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.