Identification of a novel actin binding site within the Dp71 dystrophin isoform.
ABSTRACT The Dp71 dystrophin isoform has recently been shown to localize to actin filament bundles in early myogenesis. We have identified an actin binding motif within Dp71 that is not found in other dystrophin isoforms. Actin overlay assays and transfection of COS-7 cells with fusion proteins of wild type and mutated Flag epitope-tagged Dp71 demonstrate that this motif is necessary and sufficient to direct localization of Dp71 to actin stress fibers. Furthermore, this localization is independent of alternative splicing which alters the C-terminus of the protein. The identification of an actin binding site suggests Dp71 may function to anchor membrane receptors to the cytoskeleton.
Article: Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: members of the nuclear DAPC associate with the nuclear matrix.[show abstract] [hide abstract]
ABSTRACT: Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, beta-sarcoglycan, beta-dystroglycan, alpha- and beta-syntrophin, alpha1- and beta-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, beta-dystroglycan, nNOS, beta-sarcoglycan, alpha/beta syntrophin, alpha1-dystrobrevin and beta-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, beta-dystroglycan and beta-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.Experimental Cell Research 11/2006; 312(16):3023-35. · 3.58 Impact Factor
Article: Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.[show abstract] [hide abstract]
ABSTRACT: 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.Thrombosis and Haemostasis 01/2006; 94(6):1203-12. · 5.04 Impact Factor
Article: Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS.[show abstract] [hide abstract]
ABSTRACT: In muscle, the absence of dystrophin alters the dystrophin-associated protein complex (DAPC), which is involved in the clustering and anchoring of signaling proteins and ion and water channels. Here we show that mice spermatozoa express only dystrophin Dp71 and utrophin Up71. The purpose of this study was to explore the effect of the absence of Dp71 on the morphology and membrane distribution of members of the DAPC, ion channels and signaling proteins of spermatozoa obtained from dystrophic mutant mdx3cv mice. Our work indicates that although the absence of Dp71 results in a dramatic decrease in beta-dystroglycan, it induces membrane redistribution and an increase in the total level of alpha-syntrophin, voltage-dependent Na+ (micro1) and K+ (Kv1.1) channels and neural nitric oxide synthase (nNOS). The short utrophin (Up71) was upregulated and redistributed in the spermatozoa of mdx3cv mice. A significant increase in abnormal flagella morphology was observed in the absence of Dp71, which was partially corrected when the plasma membrane was eliminated by detergent treatment. Our observations point to a new phenotype associated with the absence of Dp71. Abnormal flagellar structure and altered distribution of ion channels and signaling proteins may be responsible for the fertility problems of mdx3cv mice.Journal of Cell Science 02/2005; 118(Pt 1):137-45. · 6.11 Impact Factor