A Mutant αII-spectrin Designed to Resist Calpain and Caspase Cleavage Questions the Functional Importance of This Process in Vivo
2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês
10.1074/jbc.m700028200
ISSN1083-351X
AutoresFleur Méary, Sylvain Métral, Chrystophe Ferreira, Dominique Eladari, Yves Colin, Marie‐Christine Lecomte, Gaël Nicolas,
Tópico(s)Connexins and lens biology
Resumoα- and β-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous αII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of αII-spectrin by these enzymes could be involved in important functions. To assess the role of αII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant αII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant αII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular αII-spectrin cleavage in either major cellular functions or in normal development. α- and β-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous αII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of αII-spectrin by these enzymes could be involved in important functions. To assess the role of αII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant αII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant αII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular αII-spectrin cleavage in either major cellular functions or in normal development. Spectrin was first identified at the inner surface of the red blood cell membrane and is well known to be the central component of the membrane skeleton, a ubiquitous and complex spectrin-actin scaffold. Spectrins are long and flexible molecules composed of two subunits, α and β, that intertwine to form α/β heterodimers. Spectrin is subsequently integrated into the membrane skeleton as (α/β)2 tetramers resulting from the self-association of α/β heterodimers (1Byers T.J. Branton D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6153-6157Crossref PubMed Scopus (310) Google Scholar). These tetramers constitute the filaments of the lattice, the nodes of which are cross-linked by short actin filaments (2Mohandas N. An X. Transfus Clin. Biol. 2006; 13: 29-30Crossref PubMed Scopus (18) Google Scholar). Each spectrin subunit is mainly composed of a succession of triple helical repeats. The spectrin-based membrane skeleton is responsible for the characteristic shape and the unique physical properties of red blood cells, such as deformability and remarkable stability to shear stress. Defects in components of this network lead to membrane fragility and are associated with various hemolytic anemias (3Delaunay J. Blood Rev. 2007; 21: 1-20Crossref PubMed Scopus (200) Google Scholar). This membrane skeleton network was identified in all nonerythroid mammalian cells with components, including spectrins, that are very similar to those found into red blood cells but in most cases are expressed by different genes. There are only two genes encodingα-spectrins. TheαI-spectrin is mainly expressed into red blood cells in association with βI. The ubiquitous αII-spectrin (also named α-fodrin) is actually considered as the major α-spectrin expressed in nonerythroid cells, and the αII/βII heterodimers are the main species described in these cells. Disruption of membrane skeletons through β-spectrin mutations was found to be responsible for the abnormal localization or the maintenance of proteins associated with spectrin-based complexes (4Ikeda Y. Dick K.A. Weatherspoon M.R. Gincel D. Armbrust K.R. Dalton J.C. Stevanin G. Durr A. Zuhlke C. Burk K. Clark H.B. Brice A. Rothstein J.D. Schut L.J. Day J.W. Ranum L.P. Nat. Genet. 2006; 38: 184-190Crossref PubMed Scopus (278) Google Scholar, 5Parkinson N.J. Olsson C.L. Hallows J.L. McKee-Johnson J. Keogh B.P. Noben-Trauth K. Kujawa S.G. Tempel B.L. Nat. Genet. 2001; 29: 61-65Crossref PubMed Scopus (104) Google Scholar, 6Komada M. Soriano P. J. Cell Biol. 2002; 156: 337-348Crossref PubMed Scopus (239) Google Scholar, 7Tang Y. Katuri V. Srinivasan R. Fogt F. Redman R. Anand G. Said A. Fishbein T. Zasloff M. Reddy E.P. Mishra B. Mishra L. Cancer Res. 2005; 65: 4228-4237Crossref PubMed Scopus (78) Google Scholar). There are no mammalian models describing αII-spectrin mutations. Defects in the unique α-spectrin ortholog to the αII-gene from vertebrates are lethal in larva in Drosophila melanogaster and Caenorhabditis elegans, arguing for the crucial role of the entire protein (8Lee J.K. Coyne R.S. Dubreuil R.R. Goldstein L.S. Branton D. J. Cell Biol. 1993; 123: 1797-1809Crossref PubMed Scopus (130) Google Scholar, 9Norman K.R. Moerman D.G. J. Cell Biol. 2002; 157: 665-677Crossref PubMed Scopus (64) Google Scholar, 10Praitis V. Ciccone E. Austin J. Dev. Biol. 2005; 283: 157-170Crossref PubMed Scopus (39) Google Scholar). αII-spectrin differs from the erythroid αI-spectrin mainly in one feature as follows: the presence of a short region consisting of 36 amino acids encoded by three exons and located in the middle part of the protein near an SH3 2The abbreviations used are: SH, Src homology; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BDP, breakdown product; MEF, murine embryonic fibroblast; PGK, phosphoglycerate kinase; ES, embryonic stem; ALLN, N-acetyl-Leu-Leu-norleucinal. 2The abbreviations used are: SH, Src homology; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BDP, breakdown product; MEF, murine embryonic fibroblast; PGK, phosphoglycerate kinase; ES, embryonic stem; ALLN, N-acetyl-Leu-Leu-norleucinal. domain. This region bears several cleavage sites for different proteases, including calpains and caspases. Moreover, it contains a binding site for calmodulin, and a calmodulin/αII-spectrin interaction was shown to regulate the cleavage by calpains and caspases (11Rotter B. Kroviarski Y. Nicolas G. Dhermy D. Lecomte M.C. Biochem. J. 2004; 378: 161-168Crossref PubMed Scopus (53) Google Scholar). We and others found that calpain cleavage is also regulated by phospho/dephosphorylation of the tyrosine residue (position 1176) located in the calpain recognition site (12Nicolas G. Fournier C.M. Galand C. Malbert-Colas L. Bournier O. Kroviarski Y. Bourgeois M. Camonis J.H. Dhermy D. Grandchamp B. Lecomte M.C. Mol. Cell. Biol. 2002; 22: 3527-3536Crossref PubMed Scopus (86) Google Scholar, 13Nedrelow J.H. Cianci C.D. Morrow J.S. J. Biol. Chem. 2003; 278: 7735-7741Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). More recently, it was shown that this region here named CCC (for calpain, caspase, calmodulin) is also targeted by proteases (e.g. Pet) secreted by pathological enteroaggregative strains of Escherichia coli (14Canizalez-Roman A. Navarro-Garcia F. Mol. Microbiol. 2003; 48: 947-958Crossref PubMed Scopus (56) Google Scholar, 15Navarro-Garcia F. Canizalez-Roman A. Sui B.Q. Nataro J.P. Azamar Y. Infect. Immun. 2004; 72: 3609-3621Crossref PubMed Scopus (60) Google Scholar). Our working hypothesis is that specific proteolysis of αII-spectrin by proteases such as calpains and caspases could be a pathway for local and global membrane skeleton reorganization that occurs during different cellular mechanisms such as cell shape regulation, cell differentiation, motility, or cytokinesis (16Simonovic M. Zhang Z. Cianci C.D. Steitz T.A. Morrow J.S. J. Biol. Chem. 2006; 281: 34333-34340Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). As an example, cells treated with the Pet protease underwent actin remodeling, which might be the consequence of αII-spectrin cutting (15Navarro-Garcia F. Canizalez-Roman A. Sui B.Q. Nataro J.P. Azamar Y. Infect. Immun. 2004; 72: 3609-3621Crossref PubMed Scopus (60) Google Scholar). Cleavage by calpains could be more physiological than cutting by caspases, which are mainly activated during the irreversible apoptotic process. However, calpains can also be overactivated in pathological conditions (17Harwood S.M. Yaqoob M.M. Allen D.A. Ann. Clin. Biochem. 2005; 42: 415-431Crossref PubMed Scopus (110) Google Scholar). It has been proposed that the αII-spectrin cleavage during apoptosis can be responsible for the membrane blebbing, which is typical in this process. To address in vivo the role of the CCC αII-spectrin region, we generated mice in which this region is deleted by using gene targeting. This is the first report concerning the description of a murine mutant for αII-spectrin. Surprisingly, homozygous mice expressing this mutant spectrin are healthy, breed normally, and present no histological anomaly, arguing against the role of this particular αII-spectrin cleavage in either major cellular functions or in normal development. Construction of the Targeting Vector—Both Spna1 and Spna2 genes encoding αI- and αII-spectrin, respectively, have similar intron-exon organizations. The main difference consists in the presence of three additional exons in the Spna2 gene, which encode the specific CCC region of αII-spectrin (Fig. 1). The first exon (24 bp), which we arbitrarily named exon "24b" (to respect the nomenclature of other exons based on intron-exon organization of Spna1 gene), encodes an 8-amino acid sequence containing the calpain cleavage site. The two following exons, exons "24c" (36 bp) and "24d" (48 bp), encode the caspase cleavage site and the calmodulin binding domain, respectively. We also named "Σ1" for the alternative spliced exon located between exons 22 and 23, which encodes a 20-amino acid sequence specific for the αIIΣ1 isoform. Our strategy consists of the elimination of these three exons using a classical knockout approach. However, the rest of the encoded mutant αII-spectrin was kept intact as the deletion should not impair the translation reading frame. To evaluate the splicing of the chimeric intron resulting from Cre recombination, a minigene model was developed. A genomic DNA portion of the murine Spna2 gene, including exon 24, chimeric intron 24/24d, and exon 25, was cloned into pcDNA3 plasmid (Invitrogen). After transfection of this construct into NIH-3T3 fibroblastic cells, we demonstrated that this chimeric intron was correctly spliced using reverse transcription-PCR (data not shown). The targeted mutation was introduced at the Spna2 locus by homologous recombination in embryonic stem (ES) cells. The targeting construct was assembled as follows. All amplifications were performed with Pfx DNA polymerase (Invitrogen). All primers were from Invitrogen or MWG Biotec. Genomic DNA (129/Sv) was amplified using primers 5′-cagtcagcctcgagggagaacc-3′ and 5′-gggggatccaaccaagggcaccacagacac-3′, and the 3.5-kbp amplified product was cloned into XhoI-BamHI-linearized pBluescript II KS plasmid (Stratagene). The resulting plasmid was linearized with BamHI and NotI, and a 3.6-kbp fragment amplified with primers 5′-gggggatccttaagttcctaagttccttaaatagataagtgttttg-3′ and 5′-ataagaatgcggccgcataacttcgtatagcatacattatacgaagttatttgaatacttggtcccctttgga-3′ (LoxP site is underlined) was subcloned (after digestion with BamHI and NotI). The resulting plasmid was linearized with NotI and SacII and a 3.5-kbp fragment amplified with primers 5′-gggggatccataacttcgtataatgtatgctatacgaagttatgcggccgcacatgagcctatgggagtcattctc-3′ (LoxP site is underlined) and 5′-tccccgcggctcgagacctgtcaagtcatgaagcccagtc-3′ was subcloned (after digestion with NotI and SacII). The resulting plasmid was linearized with BamHI to receive a 2.1-kbp "floxed" PGK promoter/hygromycin resistance cassette (a generous gift from Marco Giovannini, INSERM U434, Paris, France). Orientation of the cassette was checked. Each cloned fragment was completely sequenced at each step. No mutation was found when compared with sequences from data bases. The 12.7-kbp recombination fragment was separated from pBluescript II KS backbone using XhoI digestion, agarose electrophoresis, gel purification QIAquick PCR purification kit, Qiagen), and then a second purification (QIAquick PCR purification kit, Qiagen). Gene Targeting in ES Cells and Generation of Chimeras—Following linearization at the XhoI site, the 12.7-kbp targeting vector was electroporated into 129/Sv-ter ES cells grown on feeder layers (Mouse Clinical Institute, Illkirch, France). After hygromycin selection (150 μg/ml), DNA of resistant clones was analyzed by Southern blot and PCR strategies to identify correctly targeted ES cells. To generate chimeras, targeted ES cells were injected into C57BL/6J host blastocysts that were transferred into foster females. Male chimeras were selected by coat color and crossed with C57BL/6J females to obtain germ line transmission of the targeted Spna2 allele. Males chimeras were then bred with 129/Sv-ter females in order to put the targeted allele on a homogeneous 129/Sv genetic background. The Cre recombination was performed by crossing mice bearing the targeted Spna2 allele with the MeuCre40 transgenic line (18Leneuve P. Colnot S. Hamard G. Francis F. Niwa-Kawakita M. Giovannini M. Holzenberger M. Nucleic Acids Res. 2003; 31: e21Crossref PubMed Scopus (52) Google Scholar). Recombined alleles were then segregated by crossing the mosaic mice with 129/Sv mice (Charles River Laboratories). In this study we focused only on the allele that recombined between the LoxP site 1 and LoxP site 3 (see Fig. 1). All experiments were performed in compliance with French laws on animal care, using relevant INSERM guidelines. Genotype Analysis—Genotyping on mouse DNA was performed using Southern blot (data not shown, available upon request) or by using a multiplex PCR to identify wild-type and Spna2 knock-out alleles. Genomic DNA (0.1–0.5 μg) was used in a 25-μl reaction that included five primers as follows: primer 1, 5′-gatctgaaagccaatgagtctcggc-3′ (forward, annealing in exon 24); primer 2, 5′-tacatagagaatggccagtcttttgac-3′ (forward, annealing in intron 24d), primer 3, 5′-gcacaactgggtaaggttcctattcc-3′ (reverse, annealing in intron 24d), primer 4, 5′-cccggcattctgcacgcttc-3′ (forward, annealing in PGKhygro cassette) and primer 5- 5′ tccatggcctccgcgaccg 3′ (reverse, annealing in PGKhygro cassette). The wild-type Spna2S allele amplifies a single amplicon (201 bp with primers 2 + 3, see Fig. 2A); the targeted Spna2 allele amplifies two amplicons (243 bp with primers 2 + 3 and 450 bp with primers 4 + 5), and the Cre recombined (LoxP site 1 with LoxP site 3) Spna2R allele amplifies one single amplicon (374 bp with primers 1 + 3, see Fig. 2A). PCR was performed as follows: 35 cycles (each cycle consisting of 20 s at 94 °C, 20 s at 65 °C, and 30 s at 72 °C) with an initial denaturation at 94 °C for 4 min, and a final elongation at 72 °C for 5 min in 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 2 mm MgCl2, 0.2 mm each dNTP, 0.18 mm each primer 1–3, 0.2 mm each primer 4–5, 0.5 unit of Taq polymerase (Invitrogen). The reaction was analyzed on 2% agarose gel containing SYBR Safe (Invitrogen). This PCR method for genotyping gave the same results as Southern blot (data not shown). Cre transgene genotyping was performed as described (19Rubera I. Poujeol C. Bertin G. Hasseine L. Counillon L. Poujeol P. Tauc M. J. Am. Soc. Nephrol. 2004; 15: 2050-2056Crossref PubMed Scopus (72) Google Scholar). Reverse Transcription—Total RNA was extracted from organs biopsies with RNA PLUS™ (Q-Biogen). The cDNA synthesis was performed in 20 μl with 2 μg of total RNA, in the presence of 0.5 mm of each dNTP, 10 ng/μl of random hexanucleotide primers, 0.1 mg/ml bovine serum albumin (BSA), 1 unit/μl RNase OUT (Invitrogen), 10 mm dithiothreitol, and 10 units/μl SuperScript™ reverse transcriptase (Invitrogen). The reaction was conducted for 65 min at 42 °C before reverse transcriptase was inactivated for 6 min at 96 °C. After reaction, cDNA was diluted with 80 μlof10mm Tris-HCl, 0.1 mm EDTA (pH 8.0). PCR amplification was performed with 2.5 μlof reverse transcriptase reaction mixture in 25 μlof20mm Tris-HCl (pH 8.4), 50 mm KCl, 2 mm MgCl2, 0.2 mm each dNTP, 0.2 mm each primer as follows: forward, 5′-gggaagcttccaccatggatctgaaagccaatgagtctcggc-3′ (annealing in exon 24), and reverse, 5′-gggctcgaggtgaaacctctgtacttcgtgtgcac-3′ (annealing in exon 25), 0.5 units of Taq polymerase (Invitrogen). PCR was performed as follows: 30 cycles (each cycle consisting of 20 s at 94 °C, 20 s at 60 °C, and 40 s at 72 °C) with an initial denaturation step at 94 °C for 5 min, and a final elongation step at 72 °C for 5 min. The reaction was analyzed on 1.5% agarose gel containing SYBR Safe (Invitrogen). Primary Murine Embryonic Fibroblasts (MEFs)—Primary fibroblast cultures were established from E13.5 embryos. The time of fertilization was determined by observation of copulation plugs, and noon of that day was defined as E0.5. Embryos were dissected from pregnant mutant (Spna2R/R) females that had been bred with mutant males or from wild-type females (Spna2S/S) that had been bred with wild-type males. The yolk sacs, heads, and internal organs were removed. Carcasses were carefully rinsed with D-PBS (Invitrogen), cut into very small pieces with fine scissors, and treated with trypsin/EDTA (Invitrogen; 4 ml per embryo) for 5 min at 37 °C under agitation. Clumps of cells were disrupted by repeated aspirations through a 5-ml pipette. The trypsin was neutralized by adding an equal volume of culture medium (Dulbecco's modified Eagle's medium (4.5 g/liter glucose, 110 mg/liter pyruvate sodium, 862 mg/liter GlutaMAX™ I) supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). After centrifugation, the cells were resuspended in culture medium and plated onto 10-cm plates. Fibroblasts were maintained at 37 °C under 5% CO2. Cells were diluted at a 1:5 ratio 24 h later on new plates, and confluence was obtained 3 days later. They were frozen in 90% fetal bovine serum with 10% Me2SO. Cells were passed every three or 4 days. We observed a growth crisis after passage 5 (approximately 2 weeks), so the study presented here was only performed with fibroblasts maintained at passage 4 maximum. When indicated, cells were preincubated with 5 μm calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal (ALLN); BioMol). Induction of Apoptosis—Apoptosis was triggered using either 1 mm H2O2 during 24 h in Dulbecco's modified Eagle's medium supplemented with 10% FBS or UV irradiation (312 nm) for 5 min. The UV irradiation was delivered by six bulbs (15 watts, T15M) on a 35 × 20 cm surface. Caspase and Calpain Cleavage Assay—Organs were submitted to an Ultraturrax rotor in the following ice-cold lysis buffer: 50 mm Hepes buffer (pH 7.5), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 10% glycerol, 1% Triton, 2 mm 4-(2-aminoethyl) benzenesulfonyl fluoride, 20 μg/ml pepstatin, 10 μg/ml bestatin. For post-mortem analysis, pieces of liver were kept at 4 °C for 48 h and were then treated as the other organs. Tritonsoluble extracts correspond to the supernatant obtained after centrifugation at 13,000 rpm (Eppendorf 5415R Centrifuge) for 20 min (4 °C) of the lysed organs previously kept on ice for 20 min. The pellets were further solubilized in the same lysis buffer containing 1% SDS and kept on ice for 20 min; insoluble extracts correspond to the supernatant obtained after centrifugation at 13,000 rpm for 20 min (4 °C) of these resuspended pellets. Extracts from MEF culture were obtained in a similar way except that samples were vigorously vortexed instead of treatment with the Ultraturrax rotor. Protein concentration was measured using the micro-BC assay protein quantification kit (Uptima) with bovine serum albumin (62.5 to 2000 ng) as standard samples. Wild-type or mutant endogenous αII-spectrin present in Triton-soluble cell lysates were submitted to exogenous recombinant proteases. Recombinant caspase 2 (catalog number SE-175, Biomol) and caspase 3 (catalog number 235417, Calbiochem, or catalog number C-1305, A. G. Scientific) were used at 1.5–1.7 units/μl of reaction in 10 mm dithiothreitol, 100 mm NaCl, 0.1% (v/v) CHAPS, and 50 mm Hepes buffer (pH 7.4) (final volume, 60 μl) for 2 (caspase 2) or 2–4 h (caspase 3) at 30 °C. Digestion with μ-calpains (catalog number C6108, Sigma) was used with 3.2 units/μl of reaction in 50 mm Tris-HCl buffer (pH 7.5), 150 mm NaCl, 2 mm MgCl2, 100 nm recombinant calmodulin for 1 h at 30 °C (final volume, 60 μl). Cleavage reactions were stopped by addition of electrophoresis sample buffer, and the proteins were analyzed by Western blot. Western Blotting—Samples were analyzed by SDS-PAGE (7% polyacrylamide gel) and transferred onto nitrocellulose membrane (Optitran®, Schleicher & Schuell) using a Tris/glycine buffer (Bio-Rad). After saturation in 10 mm phosphate buffer (pH 7.5), 150 mm NaCl, 0.05% Tween 20 (PBST), and 5% (w/v) nonfat-milk, blots were probed with specific antibodies. Antibodies used are as follows: immunopurified anti-SH3 domain of αII-spectrin (10 ng/ml (12Nicolas G. Fournier C.M. Galand C. Malbert-Colas L. Bournier O. Kroviarski Y. Bourgeois M. Camonis J.H. Dhermy D. Grandchamp B. Lecomte M.C. Mol. Cell. Biol. 2002; 22: 3527-3536Crossref PubMed Scopus (86) Google Scholar)); anti-αII-spectrin monoclonal antibody (50 ng/ml, FG 6090 antibody, clone AA6; BioHit); immunopurified anti-CCC region obtained by immunization of rabbits against the NH2-Cys-Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn-Ser-Ile-Lys-COOH peptide coupled to keyhole limpet hemocyanin (1:500 dilution; Covalab). The antibodies against the mutant αII-spectrin were obtained by immunization of rabbits with NH2-Ala-Val-Gln-Gln-Gln-Glu-Leu-Asn-Glu-Arg-COOH peptide coupled to keyhole limpet hemocyanin (200 ng/ml; DB-BioRun). Immunopurifications were performed against the immunization peptide or recombinant αII-spectrin peptide with HiTrap column (Amersham Biosciences). Immunocomplexes with immunopurified antibodies against mutant αII-spectrin were performed in 50 mm Tris buffer (pH 7.2), 150 mm NaCl, 10 mm MgCl2, 10 mm CaCl2. Other immunocomplexes were formed in PBST with or without 5% (w/v) nonfat milk. Detections were performed with either anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase (Nordic Immunological Laboratories) using the Supersignal West Pico chemiluminescence substrate (Pierce) and Gel Doc™ system (Bio-Rad). Immunohistochemistry on MEF—Cells cultured on glass slides were fixed in 4% paraformaldehyde for 20 min and washed in PBS. Free aldehyde groups were blocked by 50 mm NH4Cl for 10 min. Slides were washed in PBS, permeabilized in 0.5% Triton for 15 min, and washed again in PBS. To reduce nonspecific binding, slides were incubated in 0.1% PBS/BSA for 15 min. They were then incubated with rabbit polyclonal anti-SH3 αII-spectrin antibody diluted at 2 μg/ml (0.1% PBS/BSA) for 1 h at room temperature. After washings (0.1% PBS/BSA), slides were incubated for 1 h at room temperature with Alexa Fluor™ 488 goat anti-rabbit IgG (1:200 dilution) and Alexa Fluor™ 568 phalloidin (1:50 dilution; Molecular Probes) diluted in 0.1% PBS/BSA. Samples were examined by confocal microscopy using a Nikon Eclipse TE300 inverted microscope equipped with a 60× oil immersion objective NA 1.4 and a D-Eclipse C1 confocal system. Immunohistochemistry on Tissue Sections—Organs were fixed in 4% formaldehyde (Carlo Erba) in PBS and embedded in paraffin, and 3-μm sections of the paraffin block were deparaffinized with EZ-DeWax™ (BioGenex). Rehydration was completed in demineralized water. Slides were then incubated in 1× Target Retrieval Solution at pH 9.0 (DakoCytomation) and heated for 40 min at 98 °C in a heated water bath. This step unmasked antigen and allowed immunostaining on formaldehyde-fixed paraffin sections, as determined in preliminary experiments (not shown). The tank containing slides was cooled at room temperature for 20 min. To reduce nonspecific binding, sections were rinsed in 0.1% PBS/BSA for 5 min and preincubated for 20 min with antibody diluent with background reducing components (DakoCytomation). The labeling procedure was as follows: affinity-purified anti-SH3 antibodies (4 μg/ml), diluted in antibody diluent with background reducing components, were applied for 1 h at room temperature. After intensive washes with 0.1% PBS/BSA, sections were incubated with a 1:200 dilution (in antibody diluent with background reducing components) of Alexa Fluor® 488 goat anti-rabbit IgG (Molecular Probes) for 30 min at room temperature, followed by washings in 0.1% PBS/BSA. Nuclei were labeled by incubation of 0.2 μg/ml propidium iodide in 0.1% PBS/BSA for 15 min. Sections were mounted with ProLong™ Gold (Molecular Probes) and then examined by confocal microscopy. We took advantage of the platform present at the Mouse Clinical Institute (Illkirch, France) to perform an extensive histological analysis on mutant males (n = 3) or females (n = 3) and control wild-type male (n = 1) and female mice (n = 1). Mice were 6 months old. Organ biopsies analyzed were the salivary glands, pancreas, stomach (trimmed after fixation), duodenum, distal ileum, proximal colon, liver (the median lobe and half of the left lateral lobe), spleen, kidney (trim before fixation), urinary bladder, adrenal glands (both), mesenteric lymph nodes, thoracic aorta, trachea, thyroid gland, esophagus, thymus, heart, entire lung, leg muscle, tongue, white adipose tissue (paragenital fat pad), brown adipose tissue, dorsal tail, footpad and snout skin, eye, Harderian gland, knee joint, brain, and hypophysis. Specific samples from males were preputial gland, right testis and epididymis (fixed in Bouin's fluid), left testis, and epididymis (fixed in neutral buffered formalin), prostate and seminal vesicles. Supplementary samples from females were ovaries, oviducts, vagina, uterus body, uterine horns, and urinary bladder. Ka/Ks Values—The Ka/Ks value is the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks), with Ks indicating the background rate of evolution. These parameters allow an estimation of the selective forces acting on a protein sequence during evolution. If a sequence should be kept intact, a Ka/Ks value lower than 1 is expected. The process is called "purifying selection." The Ka/Ks analysis was performed using DnaSP software using cDNA from exons 23 to 27 and protein sequences available from Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, and Gallus gallus. Analysis of the Selection Pressure on the Central Domain of the αII-spectrin Using the KA/KS Evaluation Method—Analysis of the CCC sequences from data bases reveals that this region is highly conserved in vertebrates but does not exist in invertebrates. To estimate the level of constraint on the CCC region, we aligned the orthologous sequences of exon 24b, 24c, and 24d from different species (Tables 1 and 2). Compared with human, the murine sequence (separated by 91 million years (20Hedges S.B. Nat. Rev. Genet. 2002; 3: 838-849Crossref PubMed Scopus (579) Google Scholar)) shows 3 nonsynonymous mutations and 12 synonymous mutations in exon 24b, 24c, and 24d with the consequence that there are only two substitutions leading to amino acid change, giving a Ka/Ks of 0.051 (Tables 1 and 2). Compared with human, the chicken sequence (separated by 310 million years (20Hedges S.B. Nat. Rev. Genet. 2002; 3: 838-849Crossref PubMed Scopus (579) Google Scholar)) shows 1 nonsynonymous mutation and 13 synonymous mutations resulting in only one substitution at the protein level and a Ka/Ks of 0.013. These data reveal that the CCC region is under a strong purifying selection. The pressure to keep intact the protein sequence suggests that the CCC region has at least one important function. It should be noted that the purifying selection is not restricted to the CCC region because Ka/Ks values <<1 were also found when flanking exons (exon 23 to exon 27) were analyzed.TABLE 1Ka/Ks values of the CCC region Alignment of the nucleotide and amino acid (in black) sequences of exons 24b, 24c, and 24d of αII-spectrin from various species. Hs, H. sapiens; Pt, P. troglodytes; Mm, M. musculus; Rn, R. norvegicus; Bt, B. taurus; Cf, C. familiaris, and Gg, G. gallus. Changes in nucleotides or amino acids (compared to the human sequences) are indicated in gray. Open table in a new tab TABLE 2Ka/Ks values of the CCC region The value of Ka/Ks is calculated and indicated for each couple of vertebrates. Open table in a new tab Generation of Mice—To assess the role of the CCC region and particularly the cleavage of αII-spectrin, we generate
Referência(s)