Dp71ab/DAPs complex composition changes during the differentiation process in PC12 cells

PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of beta-dystroglycan, alpha1-syntrophin, alpha1-, and beta-dystrobrevins. We have established that Dp71ab forms a complex with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and alpha-, beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation.     

Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

We have adopted the PC12 cell line as in vitro cell model for studying Dp71 function in neuronal cells. These cells express a cytoplasmic (Dp71f) and a nuclear (Dp71d) isoform of Dp71 as well as various dystrophin-associated proteins (DAPs). In this study, we revealed by confocal microscopy analysis and Western blotting evaluation of cell fractions the presence of different DAPs (β-dystroglycan, β-dystrobrevin, ε-sarcoglycan and γ1-syntrophin) in the nucleus of PC12 cells. Furthermore, we established by immunoprecipitation assays that Dp71d and the DAPs form a dystrophin-associated protein complex (DAPC) in the nucleus. Interestingly, depletion of Dp71 by antisense treatment (antisense-Dp71 cells) provoked a drastic reduction of nuclear DAPs, which indicates that Dp71d is critical for DAPs stability within the nucleus. Although Up71, the utrophin gene product homologous to Dp71, exhibited increased expression in the antisense-Dp71 cells, its scarce nuclear levels makes unlikely that could compensate for Dp71 nuclear deficiency.     

Current knowledge of dystrophin and dystrophin-associated proteins in the retina

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.     

Comparative distribution of short dystrophin superfamily products in various guinea pig spermatozoa domains

In this study, the presence and cellular distribution of dystrophin family products (i.e. Dp71d, Dp71f-like protein and dystrobrevin) was examined by indirect immunofluorescence and Western blotting in guinea pig spermatozoa. Two dystrophin-associated proteins, beta-dystroglycan and alpha-syntrophin, and nNOS a protein frequently associated with alpha-syntrophin, were determined. In spermatozoa lacking plasma membrane and acrosome, Dp71f-like protein was found in the postacrosomal perinuclear theca and also in the middle piece of the flagellum. In the flagellum, Dp71f-like protein is localized together with alpha-syntrophin and nNOS. Dp71d was present in the plasma membrane of the middle piece with beta-dystroglycan, alpha-syntrophin and nNOS. Dp71d was also present in plasma membrane of the post acrosomal region, but only with nNOS. Finally, dystrobrevin was located all along skeletal flagellum structures and in the subacrosomal hemisphere of the perinuclear theca. This distinct and complementary distribution in various domains of spermatozoa may reveal a specific function for each short dystrophin family product, in the stabilization of the domains where they are located.     

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

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.     

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.     

Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein

Even though the Duchenne muscular dystrophy (DMD) gene product Dystrophin Dp71d is involved in various key cellular processes through its role as a scaffold for structural and signalling proteins at the plasma membrane as well as the nuclear envelope, its subcellular trafficking is poorly understood. Here we map the nuclear import and export signals of Dp71d by truncation and point mutant analysis, showing for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1. Binding was confirmed in cells using pull-downs, while in vitro binding assays showed direct, high affinity (apparent dissociation coefficient of c. 0.25 nM) binding of Dp71d to IMPα/β. Interestingly, treatment of cells with the microtubule depolymerizing reagent nocodazole or the dynein inhibitor EHNA both decreased Dp71d nuclear localization, implying that Dp71d nuclear import may be facilitated by microtubules and the motor protein dynein. The role of Dp71d in the nucleus appears to relate in part to interaction with the nuclear envelope protein emerin, and maintenance of the integrity of the nuclear architecture. The clear implication is that Dp71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.     

Dystrophin Dp71: The Smallest but Multifunctional Product of the Duchenne Muscular Dystrophy Gene

Dystrophin Dp71 is expressed in all tissues, with the exception of skeletal muscle, and is the main Duchenne muscular dystrophy (DMD) gene product in brain. As full-length dystrophin does in skeletal muscle, Dp71 associates with dystroglycans, sarcoglycans, dystrobrevins, syntrophins, and accessory proteins to form the dystrophin-associated protein complex (DAPC) in non-muscle tissues. Although it has been nearly 20 years since the discovery of Dp71, its study has become relevant only recently due to its direct involvement with the two main DMD non-muscular phenotypes: cognitive impairment and abnormal retinal physiology. In this review, we describe the historical background of Dp71 and the experimental models developed for its study. Additionally, we present and discuss the experimental evidence supporting the participation of Dp71 in different cellular processes, including cell adhesion, water homeostasis, cell division, and nuclear architecture. The functional diversity of Dp71 is attributed to the formation of Dp71-containing DAPC in numerous cell types and different subcellular compartments, including in plasma membrane and nucleus, as well as to the capability of Dp71-containing DAPC to work as the scaffold for proper clustering and anchoring of structural and signaling proteins to the plasma membrane and of nuclear envelope proteins to the inner nuclear membrane.     

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.     

Subcellular localization of components of the dystrophin glycoprotein complex in cultured retinal muller glial cells

The dystrophin glycoprotein complex (DGC) is a membrane-associated protein complex binding extracellular matrix (ECM) molecules, such as laminin and forming a bridge towards the cytoskeleton. The molecular composition of the DGC is cell type dependent and it is involved in cell adhesion and motility. Here we present immunocytochemical localization of beta-dystroglycan, the central member of the DGC, utrophin and Dp71f, the spliced 71 kDa dystrophin protein product of the DMD gene, in cultured retinal Muller glial cells. It is shown that beta-dystroglycan and utrophin are colocalized in clusters in all parts of Muller cells including the lamellipodium and leading edge of migrating cells. As a contrast, Dp71f labels are distinct from beta-dystroglycan and confined to the perinuclear cytoplasm of Muller cells indicating that Dp71f is not a member of the DGC in cultured Muller cells.     

Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin-dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells

We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization.     

Expression of the skeletal muscle dystrophin-dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto-Kakizaki rat

The inability of insulin to stimulate glucose metabolism in skeletal muscle fibres is a classic characteristic of type 2 diabetes. Using the non-obese Goto-Kakizaki rat as an established animal model of this type of diabetes, sucrose gradient centrifugation studies were performed and confirmed the abnormal subcellular location of the glucose transporter GLUT4. In addition, this analysis revealed an unexpected drastic reduction in the surface membrane marker beta-dystroglycan, a dystrophin-associated glycoprotein. Based on this finding, a comprehensive immunoblotting survey was conducted which showed a dramatic decrease in the Dp427 isoform of dystrophin and the alpha/beta-dystroglycan subcomplex, but not in laminin, sarcoglycans, dystrobrevin, and excitation-contraction-relaxation cycle elements. Thus, the backbone of the trans-sarcolemmal linkage between the extracellular matrix and the actin membrane cytoskeleton might be structurally impaired in diabetic fibres. Immunohistochemical studies revealed that the reduction in the dystrophin-dystroglycan complex does not induce obvious signs of muscle pathology, and is neither universal in all fibres, nor fibre-type specific. Most importantly, the expression of alpha-syntrophin and the syntrophin-associated neuronal isoform of nitric oxide synthase, nNOS, was demonstrated to be severely reduced in diabetic fibres. The loss of the dystrophin-dystroglycan complex and the syntrophin-nNOS complex in selected fibres suggests a weakening of the sarcolemma, abnormal signalling and probably a decreased cytoprotective mechanism in diabetes. Impaired anchoring of the cortical actin cytoskeleton via dystrophin might interfere with the proper recruitment of the glucose transporter to the surface membrane, following stimulation by insulin or muscle contraction. This may, at least partially, be responsible for the insulin resistance in diabetic skeletal muscles.     

Characterization of the transmembrane molecular architecture of the dystroglycan complex in schwann cells

We have demonstrated previously 1) that the dystroglycan complex, but not the sarcoglycan complex, is expressed in peripheral nerve, and 2) that alpha-dystroglycan is an extracellular laminin-2-binding protein anchored to beta-dystroglycan in the Schwann cell membrane. In the present study, we investigated the transmembrane molecular architecture of the dystroglycan complex in Schwann cells. The cytoplasmic domain of beta-dystroglycan was co-localized with Dp116, the Schwann cell-specific isoform of dystrophin, in the abaxonal Schwann cell cytoplasm adjacent to the outer membrane. beta-dystroglycan bound to Dp116 mainly via the 15 C-terminal amino acids of its cytoplasmic domain, but these amino acids were not solely responsible for the interaction of these two proteins. Interestingly, the beta-dystroglycan-precipitating antibody precipitated only a small fraction of alpha-dystroglycan and did not precipitate laminin and Dp116 from the peripheral nerve extracts. Our results indicate 1) that Dp116 is a component of the submembranous cytoskeletal system that anchors the dystroglycan complex in Schwann cells, and 2) that the dystroglycan complex in Schwann cells is fragile compared with that in striated muscle cells. We propose that this fragility may be attributable to the absence of the sarcoglycan complex in Schwann cells.     

PKCeta is localized in the Golgi, ER and nuclear envelope and translocates to the nuclear envelope upon PMA activation and serum-starvation: C1b domain and the pseudosubstrate containing fragment target PKCeta to the Golgi and the nuclear envelope

Protein kinase C (PKC) represents a family of serin/threonine kinases, playing a central role in the regulation of cell growth, differentiation and transformation. These enzymes differ in their primary structure, biochemical properties, tissue distribution and subcellular localization. The specific cellular functions of PKC isoforms are largely controlled by their localization. PKCeta, a member of the novel subfamily, is expressed predominantly in epithelial tissues. However, not much is known with respect to its mechanism of activation and regulation. Our recent studies suggest its role in cell cycle control. Here we show that PKCeta is localized at the Golgi apparatus, ER and the nuclear envelope. Furthermore, using GFP-fusion proteins of the different functional domains of PKCeta we deciphered the specific structural domains of the protein responsible for its apparent localization. We show that the cysteine-rich repeat C1b is responsible for its Golgi localization, while for its presence at the ER/nuclear envelope the pseudosubstrate containing fragment coupled to the C1 domain is required. In response to short-term activation by PMA we show translocation of PKCeta to the plasma membrane and the nuclear envelope. We demonstrate that the C1b is sufficient for its translocation to the plasma membrane. Interestingly, accumulation of PKCeta at the nuclear envelope also occurred in response to serum-starvation. It should be noted that interaction of PKCeta with the cyclin E/Cdk2 complex at the perinuclear region was recently reported by us in response to serum-starvation. Thus, our studies demonstrate translocation of PKCeta to the nuclear envelope, and suggest that the spatial regulation of PKCeta could be important for its cellular functions including effects on cell cycle control and involvement in tumor promotion.     

Acute pathophysiological effects of muscle-expressed Dp71 transgene on normal and dystrophic mouse muscle.

products of the dystrophin gene range from the 427-kDa full-length dystrophin to the 70.8-kDa Dp71. Dp427 is expressed in skeletal muscle, where it links the actin cytoskeleton with the extracellular matrix via a complex of dystrophin-associated proteins (DAPs). Dystrophin deficiency disrupts the DAP complex and causes muscular dystrophy in humans and the mdx mouse. Dp71, the major nonmuscle product, consists of the COOH-terminal part of dystrophin, including the binding site for the DAP complex but lacks binding sites for microfilaments. Dp71 transgene (Dp71tg) expressed in mdx muscle restores the DAP complex but does not prevent muscle degeneration. In wild-type (WT) mouse muscle, Dp71tg causes a mild muscular dystrophy. In this study, we tested, using isolated extensor digitorum longus muscles, whether Dp71tg exerts acute influences on force generation and sarcolemmal stress resistance. In WT muscles, there was no effect on isometric twitch and tetanic force generation, but with a cytomegalovirus promotor-driven transgene, contraction with stretch led to sarcolemmal ruptures and irreversible loss of tension. In MDX muscle, Dp71tg reduced twitch and tetanic tension but did not aggravate sarcolemmal fragility. The adverse effects of Dp71 in muscle are probably due to its competition with dystrophin and utrophin (in MDX muscle) for binding to the DAP complex.     

Neuronal nitric oxide synthase localizes through multiple structural motifs to the sarcolemma in mouse myotubes

In skeletal muscle, neuronal nitric oxide synthase is localized at the sarcolemma in association with the dystrophin glycoprotein complex (DGC). The nNOS N-terminal 231 amino acids comprise a PDZ domain (residues 1-100) and a beta-hairpin finger loop (residues 101-130) which binds alpha-syntrophin located in the DGC. Endogenous nNOS and GFP-tagged nNOS localize to the sarcolemma in mouse C2C12 myotubes. Expression of GFP-tagged nNOS domains in C2C12 myotubes reveals that the PDZ domain and the beta-hairpin finger loop of nNOS are independently capable of localizing to the sarcolemma of C2C12 myotubes. Binding studies indicate that alpha-syntrophin binds only to the beta-hairpin finger loop and not the PDZ domain of nNOS. nNOS may bind to proteins in addition to alpha-syntrophin at muscle sarcolemma.     

Myosin‐1C uses a novel phosphoinositide‐dependent pathway for nuclear localization

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin‐dependent motor protein Myosin‐1C (Myo1C) resembles the diffusion–retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.     

Dystroglycan contributes to the formation of multiple dystrophin-like complexes in brain

In muscle, dystrophin anchors a complex of proteins at the cell surface which includes alpha-dystroglycan, beta-dystroglycan, syntrophins and dystrobrevins. Mutations in the dystrophin gene lead to muscular dystrophy and mental retardation. In contrast to muscle, little is known about the localization and the molecular interactions of dystrophin and dystrophin associated proteins (DAPs) in brain. In the present study, we show that alpha-dystroglycan and dystrophin are localized to large neurones in cerebral cortex, hippocampus, cerebellum and spinal cord. Furthermore, we show that dystroglycan is a member of three distinct dystrophin-containing complexes. Two of these complexes contain syntrophin and both dystrophin and syntrophin are enriched in post-synaptic densities. These data suggest that dystrophin and DAPs may have a role in the organization of CNS synapses. Interestingly, the enrichment for syntrophin in post-synaptic densities is not affected in mice mutant for all dystrophin isoforms. Thus in the brain, unlike in muscle, the association of syntrophin with dystrophin is not crucial for the DAP complex which suggests that it may be associated with other proteins.     

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

Background  

The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene.