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• W2068754251 abstract "We previously showed that alternatively spliced ankyrins-G, the Ank3 gene products, are expressed in skeletal muscle and localize to the postsynaptic folds and to the sarcoplasmic reticulum. Here we report the molecular cloning, tissue expression, and subcellular targeting of AnkG107, a novel ankyrin-G from rat skeletal muscle. AnkG107 lacks the entire ANK repeat domain and contains a 76-residue sequence near the COOH terminus. This sequence shares homology with COOH-terminal sequences of ankyrins-R and ankyrins-B, including the muscle-specific skAnk1. Despite widespread tissue expression of Ank3, the 76-residue sequence is predominantly detected in transcripts of skeletal muscle and heart, including both major 8- and 5.6-kb mRNAs of skeletal muscle. In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and cross-reacted with three endogenous ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitrotranslated AnkG107. In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged AnkG107 transfected in primary cultures of rat myotubes was targeted to the plasma membrane. Deletion of the 76-residue insert resulted in additional cytoplasmic labeling suggestive of a reduced stability of AnkG107 at the membrane. Recruitment of the COOH-terminal domain to the membrane was much less efficient but still possible only in the presence of the 76-residue insert. We conclude that the 76-residue sequence contributes to the localization and is essential to the stabilization of AnkG107 at the membrane. These results suggest that tissue-dependent and developmentally regulated alternative processing of ankyrins generates isoforms with distinct sequences, potentially involved in specific protein-protein interactions during differentiation of the sarcolemma and, in particular, of the postsynaptic membrane. We previously showed that alternatively spliced ankyrins-G, the Ank3 gene products, are expressed in skeletal muscle and localize to the postsynaptic folds and to the sarcoplasmic reticulum. Here we report the molecular cloning, tissue expression, and subcellular targeting of AnkG107, a novel ankyrin-G from rat skeletal muscle. AnkG107 lacks the entire ANK repeat domain and contains a 76-residue sequence near the COOH terminus. This sequence shares homology with COOH-terminal sequences of ankyrins-R and ankyrins-B, including the muscle-specific skAnk1. Despite widespread tissue expression of Ank3, the 76-residue sequence is predominantly detected in transcripts of skeletal muscle and heart, including both major 8- and 5.6-kb mRNAs of skeletal muscle. In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and cross-reacted with three endogenous ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitrotranslated AnkG107. In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged AnkG107 transfected in primary cultures of rat myotubes was targeted to the plasma membrane. Deletion of the 76-residue insert resulted in additional cytoplasmic labeling suggestive of a reduced stability of AnkG107 at the membrane. Recruitment of the COOH-terminal domain to the membrane was much less efficient but still possible only in the presence of the 76-residue insert. We conclude that the 76-residue sequence contributes to the localization and is essential to the stabilization of AnkG107 at the membrane. These results suggest that tissue-dependent and developmentally regulated alternative processing of ankyrins generates isoforms with distinct sequences, potentially involved in specific protein-protein interactions during differentiation of the sarcolemma and, in particular, of the postsynaptic membrane. green fluorescent protein enhanced GFP sarcoplasmic/endoplasmic Ca2+-ATPase isoform 1 extensor digitorum longus amino acid(s) Ankyrins, the peripheral proteins that link integral membrane proteins to spectrin, are involved in the selective accumulation and local restriction of ion channels and cell adhesion molecules in specialized membrane domains (1.Bennett V. Baines A.J. Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (797) Google Scholar, 2.Beck K.A. Nelson W.J. Am. J. Physiol. (Tokyo). 1996; 270: C1263-C1270Crossref PubMed Google Scholar, 3.De Matteis M.A. Morrow J.S. J. Cell Sci. 2000; 113: 2331-2343Crossref PubMed Google Scholar). Ankyrins form a diverse family of modular polypeptides resulting from the expression of at least three genes, designated Ank1,Ank2, and Ank3 in rodents (ANK1,ANK2, and ANK3 in humans), and from tissue-specific alternative splicing of their products (ankyrins-R, ankyrins-B, and ankyrins-G, respectively) that display different subcellular localization (4.Lambert S. Bennett V. Curr. Top. Membr. 1996; 43: 129-145Crossref Scopus (6) Google Scholar). Most isoforms are composed of two highly conserved, membrane-binding (NH2-terminal) and spectrin-binding domains, and one variable COOH-terminal domain. The membrane-binding domain is composed of 24 tandem copies of 33-residue ANK repeats that provide sites of protein-protein interaction in numerous proteins (5.Bork P. Proteins. 1993; 17: 363-374Crossref PubMed Scopus (446) Google Scholar) and bind to the cytoplasmic domains of most ankyrin-associated integral proteins (6.Davis J.Q. Bennett V. J. Biol. Chem. 1990; 265: 17252-17256Abstract Full Text PDF PubMed Google Scholar, 7.Srinivasan Y. Lewallen M. Angelides K.J. J. Biol. Chem. 1992; 267: 7483-7489Abstract Full Text PDF PubMed Google Scholar, 8.Davis J.Q. McLaughlin T. Bennett V. J. Cell Biol. 1993; 121: 121-133Crossref PubMed Scopus (148) Google Scholar, 9.Morgans C.W. Kopito R.R. J. Cell Sci. 1993; 105: 1137-1142PubMed Google Scholar, 10.Michaely P. Bennett V. J. Biol. Chem. 1995; 270: 22050-22057Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11.Michaely P. Bennett V. J. Biol. Chem. 1995; 270: 31298-31302Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12.Thevananther S. Kolli A.H. Devarajan P. J. Biol. Chem. 1998; 273: 23952-23958Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Accumulating evidence shows the existence of “truncated” ankyrins, lacking a part or the totality of the membrane-binding and/or COOH-terminal domains (13.Kordeli E. Lambert S. Bennett V. J. Biol. Chem. 1995; 270: 2352-2359Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 14.Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar, 15.Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar, 16.Hoock T.C. Peters L.L. Lux S.E. J. Cell Biol. 1997; 136: 1059-1070Crossref PubMed Scopus (67) Google Scholar). Despite the lack of the membrane-binding domain, these isoforms appear associated with intracellular membrane compartments (14.Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar, 16.Hoock T.C. Peters L.L. Lux S.E. J. Cell Biol. 1997; 136: 1059-1070Crossref PubMed Scopus (67) Google Scholar). Among ankyrin genes, Ank3 shows a broad tissue expression, including kidney (14.Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar) and the nervous system (13.Kordeli E. Lambert S. Bennett V. J. Biol. Chem. 1995; 270: 2352-2359Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), where it was first identified. Tissue-specific alternative processing of Ank3transcripts results in distinct ankyrin-G isoforms with presumably related but distinct functions. The largest 480- and 270-kDa ankyrin-G isoforms are specifically expressed in neurons, where they are targeted to the nodes of Ranvier and initial axonal segments (13.Kordeli E. Lambert S. Bennett V. J. Biol. Chem. 1995; 270: 2352-2359Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 17.Zhang X. Bennett V. J. Biol. Chem. 1996; 271: 31391-31398Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18.Lambert S. Davis J.Q. Bennett V. J. Neurosci. 1997; 17: 7025-7036Crossref PubMed Google Scholar, 19.Zhang X. Bennett V. J. Cell Biol. 1998; 142: 1571-1581Crossref PubMed Scopus (102) Google Scholar). These isoforms contain extended “tail” sequences between the spectrin-binding and COOH-terminal domains. Ankyrins-G expressed in tissues other than brain lack the tail domain, and their molecular masses range from 100 to 220 kDa. Currently, cloned ankyrins-G of the latter category include: (i) epithelial mouse Ank3 polypeptides that display a polarized plasma membrane localization or a cytoplasmic distribution depending on the presence or lack of the ANK repeat domain (Ref. 14.Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar; Ank3–7kb and Ank3–5kb, respectively); (ii) AnkG190, a kidney- and lung-specific protein that contains ANK repeats and associates with Na,K-ATPase at the lateral plasma membrane of epithelial cells (12.Thevananther S. Kolli A.H. Devarajan P. J. Biol. Chem. 1998; 273: 23952-23958Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar); (iii) AnkG119, an isoform with a truncated ANK repeat domain and a very short distinct COOH-terminal domain, that binds β spectrin and associates with the Golgi apparatus and trans-Golgi network in epithelial cells (15.Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar); (iv) two short 100- and 120-kDa isoforms that lack the ANK repeat domain and associate with lysosomes in macrophages (16.Hoock T.C. Peters L.L. Lux S.E. J. Cell Biol. 1997; 136: 1059-1070Crossref PubMed Scopus (67) Google Scholar). In skeletal muscle fibers, assembly of specialized membrane domains is a functional requirement, both at the cell surface (i.e. the postsynaptic membrane and the costameres) and in the cytoplasm, where Ca2+-regulated excitation-contraction coupling occurs. In this tissue, multiple ankyrins are expressed by all three genes (15.Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar,20.Moon R.T. Ngai J. Wold B.J. Lazarides E. J. Cell Biol. 1985; 100: 152-160Crossref PubMed Scopus (27) Google Scholar, 21.Birkenmeier C.S. White R.A. Peters L.L. Hall E.J. Lux S.E. Barker J.E. J. Biol. Chem. 1993; 268: 9533-9540Abstract Full Text PDF PubMed Google Scholar, 22.Zhou D. Birkenmeier C.S. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar, 23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar, 24.Tuvia S. Buhusi M. Davis L. Reedy M. Bennett V. J. Cell Biol. 1999; 147: 995-1008Crossref PubMed Scopus (109) Google Scholar) and localize to several membrane sites, including the postsynaptic membrane (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar, 25.Flucher B.E. Daniels M.P. Neuron. 1989; 3: 163-175Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 26.Wood S.J. Slater C.R. J. Cell Biol. 1998; 140: 675-684Crossref PubMed Scopus (93) Google Scholar), the costameres (27.Nelson W.J. Lazarides E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3292-3296Crossref PubMed Scopus (69) Google Scholar), the triads (28.Flucher B.E. Morton M.E. Froehner S.C. Daniels M.P. Neuron. 1990; 5: 339-351Abstract Full Text PDF PubMed Scopus (72) Google Scholar), and the nonjunctional sarcoplasmic reticulum (22.Zhou D. Birkenmeier C.S. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar, 23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar) (Table I). Interestingly, the lack of Ank2 gene products in skeletal muscle fibers and cardiomyocytes of ankyrin-B(−/−) mice resulted in a congenital myopathy, abnormal properties of cardiac Na+ channels, and dramatic alterations in intracellular localization of Ca2+homeostasis proteins, namely the Ca2+-ATPase (SERCA) and the ryanodine receptors (24.Tuvia S. Buhusi M. Davis L. Reedy M. Bennett V. J. Cell Biol. 1999; 147: 995-1008Crossref PubMed Scopus (109) Google Scholar, 29.Chauhan V.S. Tuvia S. Buhusi M. Bennett V. Grant A.O. Circ. Res. 2000; 86: 441-447Crossref PubMed Scopus (89) Google Scholar). Ank1 and Ank3gene products still expressed in these mice cannot rescue ankyrin-B(−/−) muscle cell, indicating that ankyrins have gene-specific functions. Taken together, these data and the diversity of ankyrin gene expression and localization suggest that ankyrins play key roles in the assembly and functioning of membrane domains in skeletal muscle fibers. However, most of these isoforms have not yet been identified at the molecular level. Currently, cloned ankyrins that are expressed in skeletal muscle include the Golgi-associated AnkG119 (15.Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar) and two small, membrane-bound Ank1gene products of 20 and 26 kDa suggested to link the sarcoplasmic reticulum to the contractile apparatus (Ref. 22.Zhou D. Birkenmeier C.S. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar; skAnk1). Our previous studies (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar) identified at least two major 8- and 5.6-kb Ank3 transcripts and one major ankyrin-G polypeptide of ∼100 kDa in rat skeletal muscle. Furthermore, ankyrins-G were localized to the troughs of the postsynaptic membrane and to the sarcoplasmic reticulum of fast-twitch, SERCA1-expressing muscle fibers.Table IExpression and subcellular distribution of ankyrins in skeletal muscleIsoformGeneSubcellular distributionAnk1, 210 kDa (WB)Ank1Sarcolemma (210 kDa) (22.Zhou D. Birkenmeier C.S. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar, 23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar, 27.Nelson W.J. Lazarides E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3292-3296Crossref PubMed Scopus (69) Google Scholar)skAnk1, 20–26 kDaPostsynaptic membrane (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar) (isoform ?)Sarcoplasmic reticulum (skAnk1) (22.Zhou D. Birkenmeier C.S. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar)Ankyrin-B, 150 kDa, 220 kDa (WB)Ank2SarcolemmaSarcoplasm, A-bands (24.Tuvia S. Buhusi M. Davis L. Reedy M. Bennett V. J. Cell Biol. 1999; 147: 995-1008Crossref PubMed Scopus (109) Google Scholar)AnkG, 100 kDa (WB)Ank3Postsynaptic membrane (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar, 26.Wood S.J. Slater C.R. J. Cell Biol. 1998; 140: 675-684Crossref PubMed Scopus (93) Google Scholar) (isoform ?)AnkG107, 130 kDa (this paper)Sarcoplasmic reticulum (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar) (isoform ?)Sarcolemma (AnkG107; this paper)Shown are the products of the three ankyrin genes, Ank1,Ank2, and Ank3, that are presently identified in skeletal muscle either by Western blot analysis (WB) or by molecular cloning in this tissue (skAnk1, AnkG107). Subcellular distribution refers to immunolocalization data obtained by isoform-specific antibodies. References and corresponding isoforms, whenever known, are shown in parentheses. Open table in a new tab Shown are the products of the three ankyrin genes, Ank1,Ank2, and Ank3, that are presently identified in skeletal muscle either by Western blot analysis (WB) or by molecular cloning in this tissue (skAnk1, AnkG107). Subcellular distribution refers to immunolocalization data obtained by isoform-specific antibodies. References and corresponding isoforms, whenever known, are shown in parentheses. Here we report the cDNA cloning and characterization of a novel ankyrin isoform from rat skeletal muscle, which we name AnkG107, based on its predicted size of 106,911 Da and homology to the known isoforms of the ankyrin-G family. AnkG107 lacks the entire membrane-binding ANK repeat domain, displays highly conserved ankyrin-G spectrin-binding and COOH-terminal domains, and contains a unique among ankyrins-G 76-residue sequence near the COOH terminus, which shows homology with corresponding ankyrin-R and ankyrin-B COOH-terminal sequences and is predominantly expressed in heart and skeletal muscle. Endogenous ankyrins-G carrying this sequence displayed developmentally regulated expression and localized to the sarcolemma and to the postsynaptic membrane. Transfection of GFP1-tagged constructs expressing either the full-length molecule or AnkG107domains in rat myotubes in culture showed that this isoform is targeted to the plasma membrane apparently via the spectrin-binding domain. Moreover, these experiments strongly suggested that the muscle-specific 76-residue sequence is required for the stabilization of AnkG107 at the membrane. All molecular procedures were carried out using standard methods (30.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). An oligo(dT) and random primed rat skeletal muscle 5′-stretch plus λgt10 cDNA library (CLONTECH, Palo Alto, CA) was double screened by plaque hybridization of nylon filters (Nytran-Plus; Schleicher & Schuell) using as probes two random primed32P-labeled (Rediprime system; Amersham Biosciences, Inc.)Ank3 cDNA fragments from the spectrin-binding (bp 229–1103) and COOH-terminal (bp 1778–2255) domains, previously amplified by RT-PCR from rat skeletal muscle total RNA (PCR A and B, respectively, in Ref. 23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar). Hybridization was performed at 65 °C overnight, and posthybridization washes were at a maximum stringency of 0.2× SSC, 65 °C. cDNA inserts from clones positive to both probes were subcloned into the plasmid vector pBluescript IISK(+) (Stratagene) and sequenced (Applied Biosystems), leading to identification of one full-length clone, λ21, that contained Ank3 sequences. Computer-assisted searches of amino acid sequence homology were performed utilizing FASTA (31.Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar) and BLAST (32.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) programs. Ank3domain-specific cDNA probes were prepared by standard PCRs using as template the rat skeletal muscle cDNA clone λ21 for the spectrin-binding domain (bp 62–793) and 76-aa insert (bp 2633–2860) and a rat brain Ank3 cDNA clone kindly provided by Dr. S. Lambert (University of Massachusetts Medical School, Worcester, MA) for the membrane-binding and COOH-terminal (corresponding to bp 2255–2921 without insert) domains. The locations of the hybridization probes are illustrated in Fig. 3B. PCR products were gel-purified (Qiagen) and 32P-labeled using random primed DNA synthesis (Rediprime system; Amersham Biosciences). The 228-bp PCR product encoding the 76-aa insert was sequenced to confirm its identity. Total RNA was isolated from adult rat hind limb skeletal muscle using the guanidinium thiocyanate/phenol/chloroform method (RNA Plus; Bioprobe Systems) and enriched in poly(A+) RNA by oligo(dT) chromatography (30.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 20 μg of partially purified poly(A+) RNA were fractionated in 0.8% formaldehyde/agarose gel and transferred to nylon filters (Nytran-plus; Schleicher & Schuell). After fixation by ultraviolet light (UV cross-linker; Stratagene), filters were hybridized with rat Ank3 domain-specific cDNA probes and washed at 68 °C with 0.2× SSC, 0.1% SDS, before autoradiography. A rat multiple tissue Northern blot (CLONTECH) was first hybridized with cDNA probe encoding the 76-aa insert and then stripped and probed with the spectrin-binding domain cDNA probe. Hybridizations were performed at 68 °C overnight, and posthybridization washes were at a maximum stringency of 0.1× SSC, 0.1% SDS, 50 °C. cDNA fragments encoding the full-length AnkG107, the spectrin-binding domain (AnkG107Sp-b; first 590 aa), and the COOH-terminal domain (AnkG107Cter; aa 591–960) were amplified by PCR using the clone λ21 as a template and primers carrying EcoRI sites. A rat brain Ank3 cDNA clone was used to PCR-amplify the COOH-terminal domain lacking the 76-aa insert (AnkG107CterΔ76aa). PCR fragments were confirmed by DNA sequencing and introduced into the EcoRI site of either pcDNA3 vector (Invitrogen), or pEGFP-N1 vector (CLONTECH) under the control of the cytomegalovirus promoter while keeping in-frame with the downstream enhanced green fluorescent protein (EGFP). The cDNA construct of the full-length AnkG107 lacking the 76-aa insert (AnkG107Δ76aa-GFP) was obtained by replacing the COOH-terminal domain-containing EcoRV-KpnI fragment of construct AnkG107-GFP with the corresponding fragment of construct AnkG107CterΔ76aa-GFP. To raise antibodies against rat skeletal muscle ankyrins-G, a cDNA fragment (bp 62–793) was amplified by PCR using the λ21 clone as template and subcloned into vector pGEX-2T (Amersham Biosciences) to generate a 53-kDa fusion protein containing the NH2-terminal portion of glutathione S-transferase and amino acids 7–250 of the AnkG107 spectrin-binding domain. The recombinant fusion protein was expressed in E. coli BL21(DE3)pLysS cells and affinity-purified using glutathione-Sepharose beads according to the manufacturer's directions (Amersham Biosciences). To avoid proteolytic fragments, the affinity-purified polypeptides were applied to polyacrylamide SDS gels, and the band containing the full-length fusion protein was cut out of the gel and injected into rabbits. The resulting antiserum (anti-ankGSpbd) was affinity-purified using fusion protein coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). Antibodies against the AnkG107 76-residue insert were raised in rabbits against two peptides corresponding to amino acid residues 864–878 and 925–939 (see Fig. 1) (Eurogentec). The peptides represented sequences not included in the region of homology with the other ankyrin genes. Specific antibodies (anti-AnkG76aa) were affinity-purified against the antigenic peptides immobilized on HiTrap N-hydroxysuccinimide (NHS)-activated columns (Amersham Biosciences). In vitrotranscription and translation were carried out in TNT-coupled rabbit reticulocyte lysate systems (Promega) according to the manufacturer's protocols, using the pcDNA3-AnkG107 construct. Products were resolved by SDS-PAGE electrophoresis and revealed by Western blot analysis. In control experiments, an aliquot was removed in the beginning of the reaction and analyzed by Western blot analysis; alternatively, in vitro translation was performed with antisense AnkG107 cDNAs. Both controls provided identical results. Primary cultures of mammalian skeletal muscle cells were initiated from neonatal myogenic cells obtained by trypsinization of muscle pieces from hind limbs of 1–3-day-old rats, as previously reported (33.Cognard C. Constantin B. Rivet-Bastide M. Imbert N. Besse C. Raymond G. Development. 1993; 117: 1153-1161Crossref PubMed Google Scholar). For 3 days following plating, cells were maintained in growth medium consisting of Ham's F-12 medium (Invitrogen) with 10% heat-inactivated horse serum (Invitrogen), 10% fetal calf serum (Invitrogen), and 1% antibiotics. Myoblasts underwent myogenesis in differentiation medium. After 48 h of culture, differentiation medium containing Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% heat-inactivated horse serum was used to promote the formation of myotubes, which occurs within 15–18 h. The various AnkG107 cDNA constructs were transfected into the myoblasts using the Effectene Reagent kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. Myoblasts were cultured for 36 h on glass coverslips (50 × 104 cells) in growth medium and then rinsed twice in serum-free medium (Opti-MEM; Invitrogen) and transfected with 1 μg of plasmid cDNA per 35-mm plastic dish. Following a 16-h incubation, the transfection medium was replaced with fresh complete growth medium. Pieces of 15-day-old and adult rat extensor digitorum longus (EDL), soleus, and sternomastoid skeletal muscles were excised, rapidly frozen in liquid nitrogen, and ground into a powder. Tissue powder was added to boiling SDS-PAGE sample buffer containing 125 mm Tris-HCl, pH 6.8, 15% SDS, 20% glycerol, and 10% β-mercaptoethanol, homogenized, and passed through a 26-gauge needle. Samples were rapidly centrifuged, and the supernatant was used in SDS-PAGE and Western blot analysis, as previously described (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar). 4-day-old myotubes in culture were washed in TBS (20 mmTris-HCl, pH 7.5, 150 mm NaCl, 2 mm EGTA, 2 mm MgCl2) and lysed on ice with cold radioimmune precipitation buffer consisting of 50 mmTris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.05% Nonidet P-40, 1% Tween 20, 1% Triton X-100, 0.1% SDS, 10% glycerol supplemented with 0.5 mm phenylmethylsulfonyl fluoride, 1% aprotinin, and 1% protease inhibitor mixture (Sigma). Homogenates of myotubes were then sonicated and analyzed by SDS-PAGE and Western blot as previously described (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar). When the same transfer membrane was probed with two antibodies, the first antibody was stripped by incubation in Tris-HCl, pH 6.8, 2% SDS, 0.1 mβ-mercaptoethanol for 1 h at 57 °C. EDL and diaphragm skeletal muscles were removed by dissection from 15-day-old and adult Sprague-Dawley rats and immediately fixed with 3% paraformaldehyde, 0.1 m phosphate buffer, pH 7.4, for 1 h at 4 °C. Fixed tissue was cut to small blocks, infused with increasing sucrose solutions (0.5–2.1 m in PBS (20 mm phosphate buffer, pH 7.5, 150 mm NaCl), and frozen in liquid nitrogen. Semithin (0.5–1-μm) cryosections of muscle fibers were immunolabeled for indirect immunofluorescence with primary antibodies diluted at 2–5 μg/ml as previously described (23.Kordeli E. Ludosky M-A. Deprette C. Frappier T. Cartaud J. J. Cell Sci. 1998; 111: 2197-2207Crossref PubMed Google Scholar). Monoclonal antibodies to the Ca2+-ATPase (SERCA1) were from Affinity Bioreagents. Fluorescein isothiocyanate- and Cy3-conjugated secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Fluorescein isothiocyanate-conjugated α-bungarotoxin (1 μg/ml; Sigma) was used to label acetylcholine receptors in the postsynaptic membrane. Micrographs were taken with a Leica DMR microscope equipped with a CCD camera (Princeton Laboratories). Images were acquired, pseudocolored, and merged using the MetaView Imaging System (Universal Imaging Corporation, West Chester, PA) and arranged using Adobe Photoshop 5.0. 4-day-old myotubes in culture were fixed in 4% paraformaldehyde in TBS for 20 min at room temperature and either directly observed for GFP fluorescence or permeabilized with 0.1% Triton X-100/TBS for 10 min and labeled for indirect immunofluorescence with primary antibodies diluted at 2–5 μg/ml. RRX-conjugated secondary antibodies were from Jackson Immunoresearch Laboratories. Samples were analyzed by confocal laser-scanning microscopy (Bio-Rad MRC 1024 ES equipped with an argon/krypton laser) using an inverted microscope (Olympus IX70, Tokyo, Japan). The relative intensities of cytoplasmic and cortical GFP fluorescence were measured and scaled on 256 levels along transversal lines crossing the xy plane of the confocal optical section. Line intensity profiles were obtained with Lasersharp Processing software (Bio-Rad) and analyzed by a ratiometric method as follows. For each ratio, the mean cortical fluorescence intensity was obtained by averaging measurements at two different intersections of the line with the periphery of the myotube. The mean intensity of the homogeneous cytoplasmic labeling was directly calculated by the software from values measured inside a square region of interest devoid of cytoplasmic clusters. For comparison, the ratio of mean cortical over mean cytoplasmic fluorescence intensities (Fcortex/Fcytoplasm) was calculated and reported o" @default.
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