Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization

Abstract

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 ± 5 and 86 ± 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1′, which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin α1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future. Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 ± 5 and 86 ± 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1′, which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin α1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future. Laminin is a self-polymerizing extracellular matrix protein implicated in survival, proliferation, migration, differentiation, and apoptosis of a variety of cell types (1Miner J.H. Yurchenco P. Annu. Rev. Cell Dev. Biol. 2004; 20: 255-284Crossref PubMed Scopus (571) Google Scholar, 2Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzman 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 (666) Google Scholar). In particular, laminin plays a pivotal role in the instruction of neural cells during both tissue development and regeneration (3Stichel C.C. Muller H.W. J. Neurocytol. 1994; 23: 615-630Crossref PubMed Scopus (43) Google Scholar, 4Luckenbill-Edds L. Brain Res. Rev. 1997; 23: 1-27Crossref PubMed Scopus (222) Google Scholar). It is well accepted that laminin signaling depends on its self-polymerization (1Miner J.H. Yurchenco P. Annu. Rev. Cell Dev. Biol. 2004; 20: 255-284Crossref PubMed Scopus (571) Google Scholar), which naturally occurs on cell membranes as long as its longest arm interacts with a membrane receptor, be it a protein, such as a β1-containing integrin or dystroglycan, or a glycolipid, such as sulfatide (5Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar). Laminin polymerization and its anchorage have been proposed to cooperate for the formation of a supramolecular network, which have unequivocally been demonstrated to involve only interactions between the LN domains at the distal ends of each short arm (5Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar, 6McKee K.K. Harrison D. Capizzi S. Yurchenco P.D. J. Biol. Chem. 2007; 282: 21437-21447Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Such network consists of a regular lattice, whose struts correspond to the size of laminin short arms (7Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (226) Google Scholar), both in matrices assembled in vivo by tumors as well as in artificial matrices thermally gelled at 3.5 mg/ml. Because laminin has an important role in determining cell fate, it is of major scientific and biotechnological interest to produce artificial laminin matrices that can mimic the polymers produced by cells in vivo. Artificial self-polymerization of laminin has been obtained in vitro either by maintaining the protein above a critical concentration (8Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar) or by providing a surface of negative lipids to assist polymerization (9Kalb E. Engel J. J. Biol. Chem. 1991; 266: 19047-19052Abstract Full Text PDF PubMed Google Scholar). More recently, we have found that diluting laminin in acidic pH led to instantaneous solution polymerization, whereas such polymers adsorbed on coverslips could induce neuritogenesis and cell migration out of explants of newborn rat brains (10Freire E. Coelho-Sampaio T. J. Biol. Chem. 2000; 275: 817-822Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Freire E. Gomes F.C. Linden R. Neto V.M. Coelho-Sampaio T. J. Cell Sci. 2002; 115: 4867-4876Crossref PubMed Scopus (76) Google Scholar). When such explants were platted onto laminin adsorbed in neutral pH, laminin in EDTA, or poly-lysine, there was absolutely no migration or axonal growth. Immunolabeling of the matrices produced at each pH with anti-laminin antibodies revealed major morphological differences between them, whereas the acidic one seemed more flat and organized, which could account for its particular signaling properties to neural cells (11Freire E. Gomes F.C. Linden R. Neto V.M. Coelho-Sampaio T. J. Cell Sci. 2002; 115: 4867-4876Crossref PubMed Scopus (76) Google Scholar). Although the molecular organization of the flat laminin mesh in basement membranes seems to be stable, there is plenty of evidence supporting the notion that the supramolecular organization of laminin can change throughout development and among tissues. In the nervous system, for instance, it has been demonstrated that laminin goes from cell-associated to scattered extracellular matrix deposits or from a punctated pattern to small or large sheet-like aggregates in developing brain (4Luckenbill-Edds L. Brain Res. Rev. 1997; 23: 1-27Crossref PubMed Scopus (222) Google Scholar, 12Zhou F.C. Brain Res. Dev. Brain Res. 1990; 55: 191-201Crossref PubMed Scopus (86) Google Scholar). Cells in culture can also assemble different networks as demonstrated for midbrain and cortical neural cells (13Garcia-Abreu J. Moura-Neto V. Cavalcante L.A. J. Neurosci. Res. 1995; 40: 471-477Crossref PubMed Scopus (77) Google Scholar, 14Freire E. Gomes F.C. Jotha-Mattos T. Neto V.M. Silva Filho F.C. Coelho-Sampaio T. J. Cell Sci. 2004; 117: 4067-4076Crossref PubMed Scopus (23) Google Scholar) or Schwann cells (15Tsiper M.V. Yurchenco P.D. J. Cell Sci. 2002; 115: 1005-1015Crossref PubMed Google Scholar). In addition, we have previously described that the laminin matrix synthesized by embryonic astrocytes (which efficiently supports neurite outgrowth) resembles the artificial matrix assembled upon laminin acidification because it is flat, organized, and closely associated to the cell surface (14Freire E. Gomes F.C. Jotha-Mattos T. Neto V.M. Silva Filho F.C. Coelho-Sampaio T. J. Cell Sci. 2004; 117: 4067-4076Crossref PubMed Scopus (23) Google Scholar). Here we investigated at the molecular level the structural properties of acidic laminin polymers obtained in vitro at low protein concentration (10-50 μg/ml). We evaluated their ultrastructure and stability toward laminin fragments and concluded that such polymers are sheet-like matrices formed by interactions among the terminal ends of the short arms, whereas the long ones are left free at an orthogonal plane. Such polymers better correspond to the natural matrices characterized so far and should be considered as a model of artificial laminin substrate in further cell biology studies, as well as in possible therapeutic usage of the protein. Materials—Natural mouse laminin 111 isolated from Engelbreth-Holm-Swarm tumor was purchased from Invitrogen, and Repel Silane was from Amersham Biosciences. N-Propyl gallate, rabbit anti-laminin (diluted 1:30), sheep anti-rabbit IgG, and Cy3-conjugated antibody (diluted 1:800) were from Sigma-Aldrich. Laminin fragments E1′ and E3 were a generous gift of Dr. Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Electron Microscopy—Laminin was diluted to a final concentration of 50 μg/ml in 20 mm sodium acetate, pH 4.0, containing 1 mm CaCl2. For freeze etching method the solution was pipetted on a Balzers support disk, rapidly frozen by impact against a copper block, cooled by liquid nitrogen to -196 °C, fractured, and etched for 20 min by raising the temperature to -100 °C. After this, the specimens were rotary-shadowed with platinum at 15 °C and further coated with carbon at 90 °C, digested in sodium hypochlorite (NaClO3) for 30 min, and collected on 200-mesh copper grids. For negative staining 5 μl of the laminin solution (50 μg/ml) was deposited on a Formvar-coated copper grid. The solution was partially removed with a filter paper, and 5 μl of uranyl acetate (2%) was added. After 1 min, the solution was completely dried with a filter paper. The specimens were examined in a Zeiss EM 900 transmission electron microscope operated at 80 kV. Atomic Force Microscopy—Laminin was diluted to 50 μg/ml in acetate buffer and pipetted onto the surface of a newly cleaved mica, fixed for 2 h with 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 m sodium cacodyate buffer (pH 7.2) for 2 h, rinsed three times in acetate buffer, dehydrated through an ethanol series, and critical point dried (Balzers CPD 050). The samples were examined in a MFP-3D atomic force microscopy (Asylum Research, Santa Barbara, CA). The images were acquired in air using intermittent contact mode. Cantilever elastic constants were obtained by the thermal noise method. For intermittent contact mode, tetrahedral-shaped cantilevers (AC240TS, Olympus, Tokyo, Japan; nominal spring constant 2 N/m) were used at a low scan rate (0.5 Hz). Phase images were acquired with 512 × 512 pixels of resolution, and image processing was performed in IGOR-PRO (Wavemetrics, Portland, OR) using a MFP-3D template developed by Asylum Research. Light Scattering Measurements—Light Scattering was measured in a QuantaMaster spectrofluorometer (Photon Technology International, Lawrenceville, NJ) at 37 °C. The wavelength of the incident light was fixed at 400 nm, and scattered light was colleted at 90 °C between 350 and 450 nm. Laminin self-polymerization was initiated by diluting the protein from a stock solution either to a final concentration of 10 μg/ml in 20 mm sodium acetate (pH 4.0) or to a final concentration of μg/ml in 20 mm (pH both containing 1 mm The was previously with Repel Silane to protein binding to the The was performed in the or in the of the laminin E1′ or E3 Laminin stock were at °C previously to 37 °C in the of the at °C, aggregates are not to in stock the in buffer (8Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar). After laminin addition, the samples were and were The were by containing buffer only. matrices were obtained by diluting the stock solution to 50 μg/ml in 0.1 m either acetate (pH 4.0) or (pH with 1 mm CaCl2. of the diluted were onto coverslips and for h at For coverslips were fixed with 4% for 3 min and rinsed three times with 0.1 m used The matrices were with in for 30 min and at °C with rabbit anti-laminin antibody diluted in The matrices were three times with and with Cy3-conjugated antibody for 2 h, at After three times with and in the coverslips were in and using a microscope of the LG4-5 of α1 and surface was by using the D. S. Sci. U. S. PubMed Scopus Google Scholar), and images were using The Scholar). The atomic for laminin 1 and 2 were obtained from the for α1 and for D. Yurchenco P.D. Hohenester E. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar, D. Talts J.F. R. Hohenester E. J. 2000; PubMed Google Scholar). The were performed by either a neutral or a for of was used to the The constants used for protein and were 20 and and the temperature was to K. of the Laminin Matrix by Electron the of the matrix obtained in acidic pH, we studied its ultrastructure by the freeze etching The protein was diluted to a final concentration of 50 μg/ml in acetate buffer (pH 4.0) and onto a support It is important to that acid-induced laminin polymerization occurs upon (10Freire E. Coelho-Sampaio T. J. Biol. Chem. 2000; 275: 817-822Abstract Full Text Full Text PDF PubMed Scopus (41) Google in the polymers are formed in solution and are in the associated of the matrix revealed a as in The left of the image seems to correspond to a of the sheet-like whereas the the are the of more one polygonal The size of the struts in the matrix previously characterized in basement membrane and in laminin was between 30 and nm (7Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (226) Google Scholar, P.D. J.C. J. 1990; PubMed Scopus Google Scholar). to the size of the short arms in the laminin molecule, to to the in natural matrices K.K. Harrison D. Capizzi S. Yurchenco P.D. J. Biol. Chem. 2007; 282: 21437-21447Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Although the sizes of the struts in were in their using We the ultrastructure of the acidic polymers by negative of a of of an the of the the of the as for the uranyl because it through the in the protein as the in the electron The of the to the struts of the mesh were by struts The size found was 21 ± 5 nm, which approximately to the size of laminin short to previously described for natural and laminin matrices (7Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (226) Google Scholar, P.D. J.C. J. 1990; PubMed Scopus Google Scholar). freeze etching and negative staining indicate that acid-induced polymers are and a with a model interactions are to the by the short arms. of Laminin by Atomic Force laminin polymers were further evaluated by atomic force The of a of using a regular of a flat surface image was by the by the Phase is to the elastic and properties of the specimens on the surface V. and P. Atomic Force for Scholar), to a in image containing to found in electron microscopy was using mode. Because laminin is more and elastic the the in the in by the image and on the of the laminin we have for of the matrix in which the could be to the to the of such were providing an of 86 ± 3 nm. to the size of the laminin long which not in the mesh be to an orthogonal to of Laminin on Laminin in the of of the long arm to the formation of the acid-induced laminin network, we have investigated addition of the E3 which contains the distal two domains of the long with laminin polymerization in The was out at 10 a concentration at which no polymerization is at neutral pH (8Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar). was as the in light in solution at 400 nm. shows the of light obtained upon laminin in acidic pH in the or in the of a of the In the of fragment E1′, which contains the distal ends of two short arms, polymerization was On the other hand, the addition of fragment E3 did not affect solution polymerization, which that interactions with the long arm are not in the acidic polymerization was also investigated in neutral buffer 10 μg/ml no in light was 2 h of and the addition of did not to of the not When the concentration of laminin was to light was a of in 2 were to which led to an instantaneous in light to the at When polymerization was in a of fragment only in light was which polymerization was in the of the level of was by the The is in with previously in the that the E3 fragment did not with laminin polymerization in neutral pH H. Yamada Y. S. Cheng Y.S. Yurchenco P.D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). can be by the that we by light which not only account the also to the final sizes of the aggregates On the other hand, of the polymers by as used a between and and could be not to in of Laminin on Laminin on laminin aggregates could not be in neutral pH at 50 we have previously demonstrated that deposits of the protein the laminin solution was the surface of a (11Freire E. Gomes F.C. Linden R. Neto V.M. Coelho-Sampaio T. J. Cell Sci. 2002; 115: 4867-4876Crossref PubMed Scopus (76) Google Scholar, 14Freire E. Gomes F.C. Jotha-Mattos T. Neto V.M. Silva Filho F.C. Coelho-Sampaio T. J. Cell Sci. 2004; 117: 4067-4076Crossref PubMed Scopus (23) Google Scholar). Such deposits were characterized by and to be different from obtained in acidic between laminin aggregates obtained in could not be performed at the level because polymers obtained in neutral pH were as aggregates in electron both using negative staining or freeze etching not network of laminin neutral pH has only been at an protein concentration (7Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (226) Google Scholar). E1′ and E3 were for their of with the formation of laminin matrices adsorbed on coverslips at each pH Laminin formed flat and polymers in acidic pH, whereas in neutral pH the polymers were and E1′ disrupted both polymers, whereas E3 only the deposits obtained in neutral It is to that E3 the large aggregates of laminin deposited in neutral pH, small aggregates on the which E3 with the final it level of protein between the of the LG4-5 of Laminin at and the acidic buffer sheet-like has described the of the two distal domains in the long arm of laminin We have used the to the electrostatic surface of the protein at and 5 shows that in neutral pH are in a for of electrostatic interaction between two of a of formed by the between the distal ends of two long arms have been among formed laminin using (8Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar). On the other hand, the pH is to the surface positive, which completely interactions between the two distal ends of the long arm of laminin not be the for laminin the long arm is by the In and are in both neutral and acidic pH acidification of pH produced only a in the light of laminin the of Yurchenco and in (8Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar), that laminin could in the of a cell membrane or other cellular it that for the formation of laminin polymers was the isolated protein. In the the described polymers were formed on the cell as important for the of on membrane (5Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar). In the naturally assembled laminin network only the short arms to a whereas the long arm in contact with a laminin receptor, the to the cell The that laminin could in a cell-free the of using artificial polymers as to study the cellular response to the In context, it has been that the artificial polymers described in to the polymers produced by cells in vivo. Natural polymers, been whereas the of the polymers produced by in vitro self-polymerization was not for that the distal ends of the long arms with each whereas such interaction did not in the natural laminin Here we that laminin in acidic buffer in a concentration as low as nm (50 μg/ml) produced a polygonal network, only by the short arms. We also that upon acidification the interaction between the distal ends of the long arms is given that the surface of the LG4-5 domains completely is that acidification of two long arms, the interaction among short arms only. of the long arm is not in a natural the laminin to a cell receptor that its distal end such as and dystroglycan, or In the interaction with the receptor the long arm and it from in the laminin On the other hand, in neutral the long arms be free to with each other and the formation of the homogeneous involving the three short arms only. In we used and to that acid-induced laminin polymers are to natural polymers produced in vivo by staining and freeze etching by electron microscopy or by atomic force microscopy a between the of matrices produced by Engelbreth-Holm-Swarm and mouse embryonic cell tumors and acid-induced the of laminin fragments in the acid-induced as in the natural fragment E3 does not with polymerization in solution nor in large aggregates used for cell the using light revealed that E3 partially polymers formed in neutral pH at protein concentration. the of E3 led to the of the large aggregates in the matrices adsorbed in neutral pH These results as a point to the that the interactions between the distal ends of the long arms that to in neutral buffer as small of the sheet-like polygonal Such interactions the formation of a mesh that can be from polymers by the light which can between protein aggregates of different The that acid-induced laminin polymers, as to laminin polymers obtained in neutral pH, in vitro the structural properties of the natural laminin matrix and that obtained in using laminin in neutral buffer to be with more and polymers can be used in vitro to study their signaling properties to as well as in vivo to study the role of laminin in to the embryonic development and in tissue It is to that laminin has been out as an substrate for both the development and regeneration of the nervous system, has been associated to the of laminin in In we have demonstrated to laminin in neutral buffer, with laminin in pH of to to the for We the of with