Research ArticleCharacterization 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
Introduction
Dystrophin, the product of the Duchenne muscular dystrophy gene (DMD) [1], is an essential component of a multi-protein complex collectively termed the dystrophin-associated protein complex (DAPC) [2]. Proteins that comprise the DAPC are structurally organized into three distinct subcomplexes: the cytoskeletal proteins dystrophin, dystrobrevins (α and β subunits) and syntrophins (α, β and γ subunits); the dystroglycans (α and β subunits); and the sarcoglycans (α, β, γ, δ and ε subunits). In skeletal muscle, the DAPC is assembled around dystrophin; this scaffold links the actin cytoskeleton to the basement membrane via the transmembrane protein β-dystroglycan and anchors the syntrophins and dystrobrevins to the muscle membrane [2], [3]. Loss of dystrophin leads to disassembly of the complex and muscle degeneration [3]. It has been shown that neuronal nitric oxide synthase (nNOS) binds to skeletal muscle syntrophin through PDZ domain interactions [4]; thus, the DAPC may also regulate signal transduction.
Many of the DAPC components found in skeletal muscle, including β- and ε-sarcoglycans, dystroglycans and syntrophins, are also expressed in other tissues [5], [6], [7], [8]. Likewise, several dystrophin isoforms are ubiquitously expressed [9], [10]. Therefore, distinct DAPC may form in different tissues implying that they may have important roles outside skeletal muscle. In fact, the characterization of different DAPCs in non-muscle tissues as well as cultured cells has been recently documented [11], [12], [13].
Dystrophin Dp71 is a DMD gene isoform produced from an internal promoter located on intron 62 [14]. As dystrophin Dp71 is the most widely expressed DMD gene product, it is expected that a number of non-muscle DAPC have this protein as a key component. In fact, the association of Dp71 with proteins of the DAPC such as β-dystroglycan, dystrobrevins and syntrophins has been reported [15], [16], [17]. Moreover, it has been evidenced that Dp71 is necessary for the anchorage and/or organization of the brain DAPC [18].
An additional complication in understanding the diversity of DAPC functions has emerged from the evidence showing that several DAPC components are present in the nuclear compartment, such as α- and β-dystrobrevins [17], [19] and α-, β- and γ1-syntrophins [20]. Moreover, by using green fluorescence protein-Dp71 protein fusions (GFP-Dp71), we have previously shown that Dp71 splicing variants containing amino acids encoded by exon 71 and/or 78 distribute predominantly in the nuclei of HeLa cells [21].
Although the evidence mentioned above raises the possibility that a Dp71-containing DAPC (Dp71-DAPC) exists in the nucleus, the biochemical characterization of such protein complex remains to be approached. To address this issue, we analyzed the expression and subcellular distribution of Dp71 splicing isoforms and DAPC components in HeLa cells, a human cell line widely used to characterize the molecular mechanisms underlying the function of nuclear proteins. Using confocal microscopy and cell fractionation analyses we showed the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophins, α1- and β-dystrobrevins and nNOS in the nuclei of HeLa cells. Moreover, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Nuclear fractionation analyses together with in situ nuclear matrix preparations showed that Dp71, β-dystroglycan, α1-dystrobrevin, β-dystrobrevin, α/β syntrophin, β-sarcoglycan and nNOS, components of the nuclear DAPC, are present in the HeLa nuclear matrix. Furthermore, we found that Dp71, β-dystroglycan and β-dystrobrevin copurified with the nuclear matrix proteins lamin B1 and actin. Our findings suggest the presence of a DAPC in the nuclei of HeLa cells, and the association of several members of this complex with the nuclear matrix may indicate their participation as nuclear scaffolding proteins.
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Cell culture
HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM), (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) neonatal serum, 25 U/ml penicillin and 25 μg/ml streptomycin (Invitrogen), plated on 100-mm-diameter dishes and maintained at 37°C in a humidified incubator with CO2 atmosphere.
RNA extraction and RT-PCR
Total RNA was isolated from confluent cell cultures using a High Pure RNA Isolation Kit (Roche Applied Science, Indianapolis, IN, USA). RNA concentration was determined spectrophotometrically at
Expression and subcellular localization of endogenous Dp71 protein isoforms in HeLa cells
Using GFP-Dp71 gene fusions, we have previously demonstrated that alternative splicing determines the nuclear or cytoplasmic localization of exogenously expressed Dp71 protein isoforms in HeLa cells [21]. Hence, to corroborate these results, we decided to analyze the expression and distribution patterns of endogenous Dp71 protein isoforms in this cell line. HeLa cells are commonly used to study the function of nuclear proteins. To differentiate between Dp71 protein variants, we utilized
Discussion
The characterization of different dystrophin-associated protein complexes (DAPCs), such as those localized in the skeletal muscle, neuromuscular junction, brain, lung, retina and kidney, has been reported [3], [11], [12], [13], [17], [32]. In addition, an increasing number of cellular functions associated with these multiple protein complexes have been proposed, such as membrane stability, cellular signaling and force transduction [7], [33], [34]. The specific function of each DAPC seems to be
Acknowledgments
The authors would like to thank Dr. Manuel Hernández for supplying anti-actin monoclonal antibody and Dr. Dereck Blake for providing us with antibodies mDp71, 2166, β-CTFP, α-CTFP and β-521. We also thank Victor Tapia for technical assistance and Dr. Raul Mena for the confocal microscopy facility. We are grateful to Dr. Angela L. Guillozet-Bongaarts for the critical revision of the manuscript. This work was supported by CONACyT, Mexico: Grant 43285-M.
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These authors made equal contributions to this study.