Role of Laminin Terminal Globular Domains in Basement Membrane Assembly
2007; Elsevier BV; Volume: 282; Issue: 29 Linguagem: Inglês
10.1074/jbc.m702963200
ISSN1083-351X
AutoresKaren K. McKee, David H. T. Harrison, Stephanie Capizzi, Peter D. Yurchenco,
Tópico(s)Cellular Mechanics and Interactions
ResumoLaminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted α1-, β1-, or γ1-LN domains or with duplicated β1- or α1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of α-, β-, and γ-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin. Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted α1-, β1-, or γ1-LN domains or with duplicated β1- or α1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of α-, β-, and γ-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin. The laminins (Lm) 2The abbreviations used are: Lm, laminin; rLm, recombinant laminin; Δ, deletion; Σ, domain substitution; SC, Schwann cell; EHS, Engelbreth-HolmSwarm; WT, wild type; HA, hemagglutinin; ECM, extracellular matrix; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; BM, basement membrane. 2The abbreviations used are: Lm, laminin; rLm, recombinant laminin; Δ, deletion; Σ, domain substitution; SC, Schwann cell; EHS, Engelbreth-HolmSwarm; WT, wild type; HA, hemagglutinin; ECM, extracellular matrix; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; BM, basement membrane. are a major family of basement membrane glycoproteins each consisting of α-, β-, and γ-subunits joined together through a coiled-coil (1Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (650) Google Scholar). The α1-, α2-, α3B-, and α5-laminins possesses globular LN domains at the N terminus of each of the three subunits. In contrast, α4-laminins possess only two short arms because of truncation of the α-subunit short arm. All laminins terminate in a group of LG domains distal to the coiled-coil. Previous studies have shown that laminin-111 (Lm-111, α1β1γ1-subunit composition) self-assembles into a polymer (2Cheng Y.S. Champliaud M.F. Burgeson R.E. Marinkovich M.P. Yurchenco P.D. J. Biol. Chem. 1997; 272: 31525-31532Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 3Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar, 4Yurchenco P.D. Cheng Y.S. Colognato H. J. 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Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). A subsequent study of different members of the laminin family led to a hypothesis that those truncated laminins lacking one or more short arms would not polymerize. More recently, evaluation of binding interactions between recombinant N-terminal laminin protein fragments by plasmon resonance spectroscopy and chemical cross-linking identified not only the proposed binding between α1-β1-, α1-γ1-, and β1-γ1-LN-LEa pairs, but also self-binding between fragments containing the α1-LN domain (7Odenthal U. Haehn S. Tunggal P. Merkl B. Schomburg D. Frie C. Paulsson M. Smyth N. J. Biol. Chem. 2004; 279: 44505-44512Abstract Full Text Full Text PDF Scopus (56) Google Scholar). This type of homologous inter-domain binding was also reported for recombinant α2-, α5-, and β2-subunit fragments and was interpreted as evidence that there was less stringency of short arm LN domain interaction required for polymerization than previously thought. Type IV collagens are the only other basement membrane components known to self-assemble into a polymer. The network-like structure of α12α2[IV] collagen, consisting of 7 S domain complexes, NC1 dimers, and lateral associations, is considered to be an important element of the architecture of nearly all basement membranes (8Ruben G.C. Yurchenco P.D. Microsc. Res. Tech. 1994; 28: 13-28Crossref PubMed Scopus (22) Google Scholar, 9Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (247) Google Scholar). Nidogen-1, a glycoprotein similar in structure to nidogen-2, binds to the γ-subunit short arm of laminin through its G3 domain, to type IV collagen through G2 and G3, and to perlecan (10Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. Chu M.L. EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (375) Google Scholar, 11Poöschl E. Mayer U. Stetefeld J. Baumgartner R. Holak T.A. Huber R. Timpl R. EMBO J. 1996; 15: 5154-5159Crossref PubMed Scopus (69) Google Scholar, 12Ries A. Gohring W. Fox J.W. Timpl R. Sasaki T. Eur. J. Biochem. 2001; 268: 5119-5128Crossref PubMed Scopus (48) Google Scholar). It was proposed that nidogen acts as a bridge between laminin and type IV collagen and as an organizer of basement membrane structure (13Timpl R. Brown J.C. BioEssays. 1996; 18: 123-132Crossref PubMed Scopus (575) Google Scholar). Most basement membranes contain more than a single laminin heterotrimer along with type IV collagens, nidogens, perlecan, and agrin. An exception is found in the basement membranes of the developing nematode embryo which lack type IV collagen until later stages and in which the laminin polymer may form the only scaffolding (14Huang C.C. Hall D.H. Hedgecock E.M. Kao G. Karantza V. Vogel B.E. Hutter H. Chisholm A.D. Yurchenco P.D. Wadsworth W.G. Development (Camb.). 2003; 130: 3343-3358Crossref PubMed Scopus (108) Google Scholar). Insights into the significance of basement membrane components and their interactions in tissues were gained through loss-of-function studies arising from mutations in mice, zebrafish, and invertebrates (reviewed in Ref. 15Yurchenco P.D. Amenta P.S. Patton B.L. Matrix Biol. 2004; 22: 521-538Crossref PubMed Scopus (285) Google Scholar). Targeted inactivation of the LAMC1 and LAMB1 genes coding for the common γ1- and β1-laminin subunits revealed that laminins are essential for the formation of several embryonic basement membranes (16Chen Z.L. Strickland S. J. Cell Biol. 2003; 163: 889-899Crossref PubMed Scopus (230) Google Scholar, 17Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. J. Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (408) Google Scholar, 18Miner J.H. Li C. Mudd J.L. Go G. Sutherland A.E. Development (Camb.). 2004; 131: 2247-2256Crossref PubMed Scopus (231) Google Scholar). In contrast, inactivation of the mouse genes for type IV collagen, nidogens, and the nidogen-binding site in the laminin γ1-subunit, although lethal by mid-embryonic development to birth, did not prevent basement membrane assembly in most tissues but instead caused structural defects and instability of basement membranes in different tissues (19Willem M. Miosge N. Halfter W. Smyth N. Jannetti I. Burghart E. Timpl R. Mayer U. Development (Camb.). 2002; 129: 2711-2722Crossref PubMed Google Scholar, 20Poöschl E. Schlotzer-Schrehardt U. Brachvogel B. Saito K. Ninomiya Y. Mayer U. Development (Camb.). 2004; 131: 1619-1628Crossref PubMed Scopus (547) Google Scholar, 21Bose K. Nischt R. Page A. Bader B.L. Paulsson M. Smyth N. J. Biol. Chem. 2006; 281: 11573-11581Abstract Full Text Full Text PDF Scopus (57) Google Scholar). Genetic evidence supporting a role of laminin polymerization was found in the phenotype of the dy2J mouse in which defective basement membranes were observed in skeletal muscle and peripheral nerve Schwann cell endoneurium (reviewed in Ref. 15Yurchenco P.D. Amenta P.S. Patton B.L. Matrix Biol. 2004; 22: 521-538Crossref PubMed Scopus (285) Google Scholar). The underlying genetic defect of dy2J is a splice-donor mutation resulting in an in-frame deletion within the α2-LN domain that has been correlated with a failure of α2-laminin polymerization (22Sunada Y. Bernier S.M. Utani A. Yamada Y. Campbell K.P. Hum. Mol. Genet. 1995; 4: 1055-1061Crossref PubMed Scopus (154) Google Scholar, 23Colognato H. Yurchenco P.D. Curr. Biol. 1999; 9: 1327-1330Abstract Full Text Full Text PDF PubMed Google Scholar). Studies on embryoid bodies, a model of early embryonic development, and cultured Schwann cells have further implicated laminin polymerization as acting in concert with anchorage as key contributors to basement membrane assembly (24Li S. Harrison D. Carbonetto S. Faössler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar, 25Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (114) Google Scholar). An understanding of the assembly mechanisms is of value not only because of the role played by basement membranes in embryonic development and the pathogenesis of several diseases, but also because manipulations of assembly to produce more stable basement membranes hold promise as a therapeutic approach to correcting basement membrane defects (26Moll J. Barzaghi P. Lin S. Bezakova G. Lochmuller H. Engvall E. Muöller U. Ruegg M.A. Nature. 2001; 413: 302-307Crossref PubMed Scopus (193) Google Scholar). In this study we have focused on the contributions of laminin domains to the assembly process. Lm-111 heterotrimers were generated by recombinant expression in human embryonic kidney 293 cells and evaluated for their ability to polymerize, interact with nidogen-1 and type IV collagen, and form a basement membrane on cultured Schwann cells. These cells, involved in laminin-dependent congenital muscular dystrophy, were chosen because it has been found that native laminin-111 assembles a basement membrane-type ECM on the cell surfaces in culture, that laminin attachment to the cells (anchorage) is substantially mediated by sulfatides, and that dystroglycan, although not required for assembly, associates with this ECM resulting in the functional readouts of Src activation and utrophin recruitment (25Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (114) Google Scholar). β1 integrins were not found to contribute to basement membrane assembly in this model. The data of this study support the hypothesis that all three LN domains are essential for laminin polymerization and formation of a basement membrane. They also reveal a requirement of the laminin LG domains for basement membrane assembly consistent with the concept that laminin becomes tethered to the cell surface through its LG domains, whereas type IV collagen and nidogen become tethered primarily to laminin. In addition, the data provide cellular evidence for the importance of nidogen serving as a bridge between the laminin and type IV collagen polymers. The wild-type cDNAs for mouse laminin α1, human β1, and human γ1 (mα1-pCIS, hβ1-pCIS, hβ1-pCEP4, hγ1-pRc/CMV2, γ1-wtCf, and α1-wtNf) have been described previously (27Yurchenco P.D. Quan Y. Colognato H. Mathus T. Harrison D. Yamada Y. O'Rear J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10189-10194Crossref PubMed Scopus (122) Google Scholar, 28Smirnov S.P. McDearmon E.L. Li S. Ervasti J.M. Tryggvason K. Yurchenco P.D. J. Biol. Chem. 2002; 277: 18928-18937Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Colognato-Pyke H. O'Rear J.J. Yamada Y. Carbonetto S. Cheng Y.S. Yurchenco P.D. J. Biol. Chem. 1995; 270: 9398-9406Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Refer to supplemental Table 1 for details of laminin constructs. Restriction enzymes, T4 DNA ligase, and calf intestinal alkaline phosphatase were obtained from New England Biolabs and Fermentas. PCRs were carried out using PlatPfx (Invitrogen) and a PTC-100 thermal cycler (MJ Research). α1-wtNm—The 5′-end of the mα1 cDNA was amplified from mα1-pCIS utilizing primers ma1p4 and ma1F21. Three subsequent overlapping PCRs with primers ha1p8, ha1p9, and ha1p6 were carried out to synthesize a 5′-fragment that contained a NotI site followed by a 5′-untranslated region, BM40 signal sequence, c-Myc epitope tag, enterokinase cleavage site, and the 5′-terminal region of mα1. The 3′-end of the mα1 was amplified with primers ma1F20 and ma1F25. Both PCR fragments were digested with NotI and BspHI. A BspHI restriction fragment from mα1-pCIS was gel-purified (UltraClean 15 DNA purification kit, MO BIO Laboratories, Inc.) and ligated into a NotI-digested DHpuro vector (described below) along with the 5′ and 3′ PCR products. The ligated material was transformed into DH5α bacteria (Invitrogen) and plated onto LB-agar plates containing 10 μg/ml ampicillin (Sigma), and resistant clones were isolated and grown in LB media, and DNA minipreps were performed with an UltraClean miniplasmid prep kit (MO BIO Laboratories, Inc.). Ligation junctions and PCR products were checked by restriction digestion and DNA sequencing. α1ΔLNNm—Nhel and BstEII-Nhel fragment were isolated from α1-wtNm. Primers ma1F90 and 050604-13 were used in a PCR of mα1-pCIS to generate a fragment which was subsequently amplified with overlapping primer ma1F35 and then 050604-11 along with 050604-13 to generate the required 5′ sequence. The final PCR product was digested with NheI and BstEII and ligated with the other two isolated RE fragments. α1ΔLN-L4bNm—A PCR fragment was produced from α1-wtNm with primers da1-1f and da1-2r and sewn together with a second fragment, generated with da1-2f and da1-1r, using da1-1f and da1-1r and digested with AflII. An AflII fragment was isolated from α1-wtNm. Both fragments were ligated into an AflII prepared α1-wtNm. α1ΔLG1-5Nf—An AflII-SacII fragment of mα1-pRCX3 (27Yurchenco P.D. Quan Y. Colognato H. Mathus T. Harrison D. Yamada Y. O'Rear J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10189-10194Crossref PubMed Scopus (122) Google Scholar) was replaced with a PCR fragment generated from the same construct but utilizing a PCR primer to introduce a new SacII site and a STOP codon after the end of the coiled-coil domain. α1ΔLG1-5Nm—An AflII-AgeI fragment from α1-wtNm was replaced with a PCR insert produced by ligating two PCR fragments. One fragment was generated with dg1 and dg2 and a second with dg3 and dg4, and both fragments were combined with dg1 and dg4. β1-wtNh—An N-terminal PCR fragment containing a hemagglutinin epitope tag (HA; Roche Applied Science) was generated from hβ1-pCIS using four successive rounds of PCR with four sense primers (hb1-1, hb1-2, hb1-3, and hb1-4) and a single antisense primer (hb1-re1) and digested with NheI and EcoRI. A 3′-end PCR fragment was generated with hb1-re4 and hb1-10 and digested with MluI and KpnI. An EcoRI-MluI fragment was purified from hβ1-pCIS and ligated along with the two PCR fragments into the expression vector pcDNA3.1/zeo+ (Invitrogen), which had earlier been digested with NheI and KpnI. β1ΔLNNh—The same approach used to generate β1-wtNh was employed, except hb1-23, hb1-25, hb1-3, hb1-4, and hb1-re5 were used to generate the N-terminal PCR fragment that was digested with NheI and AatII. Also, an AatII-MluI fragment isolated from hβ1-pCIS was used. Both the C-terminal PCR fragment and prepared vector, used in constructing β1-wtNh, were utilized. β1ΔLN-LEaNh—The same approach used to generate β1-wtNh was employed, except hb1-28, hb1-30, hb1-3, hb1-4, and hb1-re7 were utilized to generate the N-terminal segment that was digested with NheI and BstEII. Likewise, a BstEII-MluI fragment was isolated from hβ1-pCIS. γ1-ΔLNCf—A NotI restriction digest fragment was isolated from γ1-wtCf as well as an ApaLI-NotI fragment. An N-terminal fragment was generated by two overlapping PCRs with gg1, gg3, and gg6 followed by digestion with NotI and ApaLI and ligation with the two isolated restriction fragments. γ1-ΔLN-LEaCf—A NotI restriction digest fragment was isolated from γ1-wtCf as well as an AflII-NotI fragment. A 5′-terminal fragment was generated by two overlapping PCRs with gg9, gg4, and gg5 followed by digestion with NotI and AflII and ligation with the two isolated restriction fragments. γ1Σa1LNCf—A PCR fragment was produced from mα1-pCIS with primers a1s-1F and a1s-2R. A second fragment was generated from γ1-wtCf with a1s-2F and a1s-1R. The two fragments were sewn together with primers als-1F and a1s-1R. The PCR fragment and γ1-wtCf were both digested with SacII and AflII. The PCR fragment was then used to replace the corresponding fragment in γ1-wtCf. γ1Σβ1LNCf—A PCR fragment was produced from hβ1-pCIS with primers b1s-1F and b1s-2R. A second fragment was generated from γ1-wtCf with b1s-2F and b1s-1R. The two fragments were ligated with primers bls-1F and b1s-1R. The PCR fragment and γ1-wtCf were both digested with SacII and BsrGI. The PCR fragment was used to replace the corresponding fragment in γ1-wtCf. γ1-N802SCf and γ1-P801QCf—To generate N802S (Asn to Ser; AAC to AGC) or P801Q (Pro to Gln; CCC to CAG), a BsiMI-AflII PCR fragment derived from ligating two overlapping PCR-generated fragments was utilized to replace the corresponding BsiMI-AflII fragment in γ1-wtCf. The 5′-fragment was generated with Nd-1f and PQ-2r or NS-2r. Likewise, the 3′-fragment was generated with PQ-2f or NS-2f and Nd-1r. Both 5′- and 3′-fragments were combined with Nd-1f and Nd-1r and digested with BsiMI and AflII. DHpuro—A vector that imparts puromycin resistance was constructed by replacing an AvrI-PciI fragment of pcDNA3.1/Hygro (Invitrogen) with a PCR fragment synthesized from pPUR (Clontech) containing the removed SV40 promoter sequence, puromycin resistance gene, an SV40 polyadenylation signal sequence, and a PciI site. Human embryonic kidney cells (HEK293) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biological), 200 mm l-glutamine, and penicillin-streptomycin (1,000 units/ml penicillin and 1,000 μg/ml streptomycin; Invitrogen). (a) Plasmids containing laminin subunits were stably transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Stable cell lines expressing recombinant laminins were supplemented with puromycin (α1 chains), Zeocin (β1 chains), and G418 (γ1 chains) at a final concentration of 1, 100, and 500 μg/ml, respectively. Immunoprecipitation, SDS-PAGE, and Western blot analysis of secreted protein was used to confirm expression of trimeric laminin in the stable cell lines. The α1-, β1-, and γ1-laminin chains were detected with antibodies specific for Myc (Roche Applied Science), HA (Roche Applied Science), and FLAG (Sigma) epitopes, respectively. Recombinant laminin was purified from media on a heparin-agarose (Sigma) column and eluted with 500 mm NaCl (in 50 mm Tris, pH 7.4, 1 mm EDTA). Heparin-eluted rLm1 was further bound to FLAG M2-agarose (Invitrogen) and eluted with 100 μg/ml FLAG peptide in wash buffer (150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA). The diluted protein was concentrated in an Amicon Ultra-15 filter (100,000, molecular weight cut-off) and dialyzed in TBS50 (90 mm NaCl, 50 mm Tris, pH 7.4, 0.125 mm EDTA). (b) A pCIS vector encoding full-length mouse nidogen-1 (gift of Rupert Timpl, Max Planck Institute for Biochemistry, Martinsried, Germany) was used to stably transfect cells. Secreted protein was purified from medium by metal chelating chromatography as described (10Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. Chu M.L. EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (375) Google Scholar). (c) Type IV collagen was extracted from lathyritic mouse EHS tumor and purified by salt fractionation and DEAE-cellulose chromatography as described (30Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (244) Google Scholar). (d) Laminin-111 was extracted with EDTA from lathyritic EHS tumor and purified by gel filtration and DEAE-Sephacel chromatography (unbound fraction) as described (3Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Laminin, type IV collagen, and nidogen-1 concentrations were determined by absorbance at 280 nm, amino acid analysis, and comparison against known standards in Coomassie Bluestained gels as described (2Cheng Y.S. Champliaud M.F. Burgeson R.E. Marinkovich M.P. Yurchenco P.D. J. Biol. Chem. 1997; 272: 31525-31532Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 3Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar, 30Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (244) Google Scholar). Proteins were solubilized in Laemmli sample buffer and evaluated by SDS-PAGE under reducing conditions on 6% acrylamide gels. Electrophoresed gels were stained with Coomassie Brilliant Blue R-250, imaged with Bio-Rad Gel Doc 2000 in bright field mode, and analyzed with Quantity One software (Bio-Rad). Aliquots (50 μl) of laminin in polymerization buffer (TBS, 1 mm CaCl2, 0.1% Triton X-100) at various concentrations were incubated at 37 °C in 0.5-ml Eppendorf tubes. Samples were centrifuged at 11,000 × g followed by dissolution of supernatant and pelleted fractions in SDS and analysis by SDS-PAGE. Similarly, increasing quantities of type IV collagen with constant laminin and nidogen (0.1 and 0.02 mg/ml, respectively) were incubated in PBS (with 0.1% Triton X-100) and analyzed as above. The Coomassie Blue-stained laminin β1 band and collagen α2 bands, which migrate separately, were used to determine the laminin and collagen amounts in the supernatant and pellet fractions. Nidogen binding to laminin was determined by immunoblotting. One μg of recombinant laminin was slot-blotted onto a polyvinylidene difluoride membrane (Bio-Rad). Following a 1-h blocking step with 5% milk in TTBS (50 mm Tris, pH 7.4, 90 mm NaCl, 0.05% Tween 20), the laminin slots were incubated with various quantities of nidogen. Bound nidogen was detected with rabbit polyclonal antibody at 5 μg/ml and anti-rabbit horseradish peroxidase (Pierce) and quantitated by chemical luminescence in dark field mode with the Gel-Doc apparatus. Schwann cells isolated from sciatic nerves from newborn Sprague-Dawley rats were the kind gift of Dr. James Salzer (New York University). These cells were expanded in culture for 10 passages and maintained in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum (Gemini Bio Products), neuroregulin (0.5 μg/ml, Sigma), forskolin (0.2 μg/ml, Sigma), 1% glutamine, and penicillin-streptomycin. Cells at passages 11-17 were treated with laminin, type IV collagen, and/or nidogen-1 after plating onto 24-well dishes (Costar) at half-confluent density. The following day, the media were changed, and the cells were incubated with laminins (20-40 μg/ml), type IV collagen (10-20 μg/ml), and/or nidogen-1 (2-8 μg/ml) at 37 °C for 1 h followed by washing and fixation. For electron microscopy, SCs cells were plated in 60-mm Permanox dishes (Nalgene, Nunc) 2 days prior to addition of ECM proteins. Schwann cells grown in the presence of extracellular proteins were rinsed three times with PBS (10 mm sodium phosphate, pH 7.4, 127 mm NaCl) and fixed in 3% paraformaldehyde for 30 min. Cultures were blocked with 5% goat serum and then stained with primary and appropriate secondary antibodies conjugated with fluorescent probes. Rabbit polyclonal antibodies specific for laminin-111 (EHS), laminin fragment E4 (β1LN-LEa), recombinant laminin α1LG4-5 (RG50), and nidogen-1 were used as described (24Li S. Harrison D. Carbonetto S. Faössler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar). EHS laminin antibody was titered in wells coated (1 μg/ml) with recombinant laminins (WTa, β1ΔLN, α1ΔLN, α1ΔLN-L4b, γ1ΔLN, and α1ΔLG1-5) and evaluated by direct enzyme-linked immunosorbent assay with serial 2-fold dilutions of antibody. The binding plots were essentially identical for all substrates except for α1ΔLN-L4b whose plot lagged by a single 2-fold dilution and whose color intensity at saturation (5 μg/ml) was decreased by <10%. This antibody (20 μg/ml) was used to compare the accumulation of different laminins on cell surfaces. Nidogen-specific rabbit antibody prepared against recombinant nidogen-1 (25Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (114) Google Scholar) was used at 3 μg/ml, and type IV collagen-specific rabbit antibody (Chemicon) was used at a 1:100 dilution. Detection was accomplished with Alexa Fluor 488 and 647 goat anti-rabbit IgG secondary antibodies (Molecular Probes) at 1:500 and 1:100 respectively, and fluorescein isothiocyanate-conjugated donkey anti-mouse IgM at 1:100 (Jackson ImmunoResearch). Slides were counterstained with DAPI and imaged as described (24Li S. Harrison D. Carbonetto S. Faössler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar). Laminin, type IV collagen, and nidogen immunofluorescence levels were quantitated from digital images (average of 9, each 1300 × 1030 pixels, 437 × 346 μm) recorded using a ×20 microscope objective with IPLab 3.7 software (Scanalytics). A segmentation range was chosen to subtract background and acellular immunofluorescence. The sum of pixels and their intensities in highlighted cellular areas of fluorescence were measured and normalized by dividing by the number of cells determined by a count of DAPI-stained nuclei for each image. Data were expressed as the mean ± S.D. of normalized summed intensities with the data analyzed by one-way analysis of variance with Holm-Sidak comparisons in SigmaPlot version 9.01 and SigmaStat version 3.1 (Systat). For Rotary shadow Pt/C replicas, laminin (25-50 μg/ml in 0.15 m ammonium bicarbonate, 60% glycerol) was sprayed onto mica disks, evacuated in a BAF500K unit (Balzers), rotary-shadowed with 0.9 nm Pt/C at an 8° angle, and backed with 8 nm carbon at a 90° angle as otherwise described (3Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Cells adherent to plastic were fixed in 0.5% glutaraldehyde and 0.2% tannic acid, transferred to modified Karnovsky’s fixative, washed with PBS, post-fixed in 1% osmium tetroxide, and prepared for electron microscopy as described (31Tsiper M.V. Yurchenco P.D. J. Cell Sci. 2002; 115: 1005-1015Crossref PubMed Google Scholar). Protein sequences of the α1-, β1-, and γ1-subunits of laminin were modified as shown in Fig. 1. The new domain nomenclature described by Aumailley et al. (1Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzm
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