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. 1988 Oct 1;107(4):1599–1610. doi: 10.1083/jcb.107.4.1599

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

PMCID: PMC2115247  PMID: 2459133

Abstract

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

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Selected References

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  1. Abrahamson D. R., Caulfield J. P. Distribution of laminin within rat and mouse renal, splenic, intestinal, and hepatic basement membranes identified after the intravenous injection of heterologous antilaminin IgG. Lab Invest. 1985 Feb;52(2):169–181. [PubMed] [Google Scholar]
  2. Abrahamson D. R., Caulfield J. P. Proteinuria and structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin G. J Exp Med. 1982 Jul 1;156(1):128–145. doi: 10.1084/jem.156.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Abrahamson D. R., Hein A., Caulfield J. P. Laminin in glomerular basement membranes of aminonucleoside nephrotic rats. Increased proteinuria induced by antilaminin immunoglobulin G. Lab Invest. 1983 Jul;49(1):38–47. [PubMed] [Google Scholar]
  4. Abrahamson D. R., Perry E. W. Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys. J Cell Biol. 1986 Dec;103(6 Pt 1):2489–2498. doi: 10.1083/jcb.103.6.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Abrahamson D. R. Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes. J Histochem Cytochem. 1986 Jul;34(7):847–853. doi: 10.1177/34.7.3519749. [DOI] [PubMed] [Google Scholar]
  6. Andujar M. B., Magloire H., Hartmann D. J., Ville G., Grimaud J. A. Early mouse molar root development: cellular changes and distribution of fibronectin, laminin and type-IV collagen. Differentiation. 1985;30(2):111–122. doi: 10.1111/j.1432-0436.1985.tb00522.x. [DOI] [PubMed] [Google Scholar]
  7. Aumailley M., Nurcombe V., Edgar D., Paulsson M., Timpl R. The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites. J Biol Chem. 1987 Aug 25;262(24):11532–11538. [PubMed] [Google Scholar]
  8. Bender B. L., Jaffe R., Carlin B., Chung A. E. Immunolocalization of entactin, a sulfated basement membrane component, in rodent tissues, and comparison with GP-2 (laminin). Am J Pathol. 1981 Jun;103(3):419–426. [PMC free article] [PubMed] [Google Scholar]
  9. Courtoy P. J., Timpl R., Farquhar M. G. Comparative distribution of laminin, type IV collagen, and fibronectin in the rat glomerulus. J Histochem Cytochem. 1982 Sep;30(9):874–886. doi: 10.1177/30.9.7130672. [DOI] [PubMed] [Google Scholar]
  10. Covault J., Cunningham J. M., Sanes J. R. Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle. J Cell Biol. 1987 Dec;105(6 Pt 1):2479–2488. doi: 10.1083/jcb.105.6.2479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dziadek M., Fujiwara S., Paulsson M., Timpl R. Immunological characterization of basement membrane types of heparan sulfate proteoglycan. EMBO J. 1985 Apr;4(4):905–912. doi: 10.1002/j.1460-2075.1985.tb03717.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dziadek M., Paulsson M., Aumailley M., Timpl R. Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor. Eur J Biochem. 1986 Dec 1;161(2):455–464. doi: 10.1111/j.1432-1033.1986.tb10466.x. [DOI] [PubMed] [Google Scholar]
  13. Dziadek M., Paulsson M., Timpl R. Identification and interaction repertoire of large forms of the basement membrane protein nidogen. EMBO J. 1985 Oct;4(10):2513–2518. doi: 10.1002/j.1460-2075.1985.tb03964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edgar D., Timpl R., Thoenen H. The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival. EMBO J. 1984 Jul;3(7):1463–1468. doi: 10.1002/j.1460-2075.1984.tb01997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engel J., Furthmayr H. Electron microscopy and other physical methods for the characterization of extracellular matrix components: laminin, fibronectin, collagen IV, collagen VI, and proteoglycans. Methods Enzymol. 1987;145:3–78. doi: 10.1016/0076-6879(87)45003-9. [DOI] [PubMed] [Google Scholar]
  16. Engel J., Odermatt E., Engel A., Madri J. A., Furthmayr H., Rohde H., Timpl R. Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J Mol Biol. 1981 Jul 25;150(1):97–120. doi: 10.1016/0022-2836(81)90326-0. [DOI] [PubMed] [Google Scholar]
  17. Fleischmajer R., Timpl R., Dziadek M., Lebwohl M. Basement membrane proteins, interstitial collagens, and fibronectin in neurofibroma. J Invest Dermatol. 1985 Jul;85(1):54–59. doi: 10.1111/1523-1747.ep12275341. [DOI] [PubMed] [Google Scholar]
  18. Foidart J. M., Bere E. W., Jr, Yaar M., Rennard S. I., Gullino M., Martin G. R., Katz S. I. Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Lab Invest. 1980 Mar;42(3):336–342. [PubMed] [Google Scholar]
  19. Gil J., Martinez-Hernandez A. The connective tissue of the rat lung: electron immunohistochemical studies. J Histochem Cytochem. 1984 Feb;32(2):230–238. doi: 10.1177/32.2.6363520. [DOI] [PubMed] [Google Scholar]
  20. Goldberg M., Escaig-Haye F. Is the lamina lucida of the basement membrane a fixation artefact? Eur J Cell Biol. 1986 Dec;42(2):365–368. [PubMed] [Google Scholar]
  21. Goodman S. L., Deutzmann R., von der Mark K. Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading. J Cell Biol. 1987 Jul;105(1):589–598. doi: 10.1083/jcb.105.1.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Graf J., Iwamoto Y., Sasaki M., Martin G. R., Kleinman H. K., Robey F. A., Yamada Y. Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding. Cell. 1987 Mar 27;48(6):989–996. doi: 10.1016/0092-8674(87)90707-0. [DOI] [PubMed] [Google Scholar]
  23. Griffiths G., Simons K., Warren G., Tokuyasu K. T. Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins. Methods Enzymol. 1983;96:466–485. doi: 10.1016/s0076-6879(83)96041-x. [DOI] [PubMed] [Google Scholar]
  24. JAKUS M. A. Further observations on the fine structure of the cornea. Invest Ophthalmol. 1962 Apr;1:202–225. [PubMed] [Google Scholar]
  25. Kanwar Y. S., Farquhar M. G. Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes. J Cell Biol. 1979 Apr;81(1):137–153. doi: 10.1083/jcb.81.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kleinman H. K., Cannon F. B., Laurie G. W., Hassell J. R., Aumailley M., Terranova V. P., Martin G. R., DuBois-Dalcq M. Biological activities of laminin. J Cell Biochem. 1985;27(4):317–325. doi: 10.1002/jcb.240270402. [DOI] [PubMed] [Google Scholar]
  27. Laurie G. W., Leblond C. P., Inoue S., Martin G. R., Chung A. Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney. Am J Anat. 1984 Apr;169(4):463–481. doi: 10.1002/aja.1001690408. [DOI] [PubMed] [Google Scholar]
  28. Laurie G. W., Leblond C. P., Martin G. R. Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes. J Cell Biol. 1982 Oct;95(1):340–344. doi: 10.1083/jcb.95.1.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lesot H., Kühl U., Mark K. Isolation of a laminin-binding protein from muscle cell membranes. EMBO J. 1983;2(6):861–865. doi: 10.1002/j.1460-2075.1983.tb01514.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Madri J. A., Roll F. J., Furthmayr H., Foidart J. M. Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney. J Cell Biol. 1980 Aug;86(2):682–687. doi: 10.1083/jcb.86.2.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martin G. R., Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57–85. doi: 10.1146/annurev.cb.03.110187.000421. [DOI] [PubMed] [Google Scholar]
  32. Martinez-Hernadez A. Electron immunohistochemistry of the extracellular matrix: an overview. Methods Enzymol. 1987;145:78–103. doi: 10.1016/0076-6879(87)45004-0. [DOI] [PubMed] [Google Scholar]
  33. Martinez-Hernandez A., Chung A. E. The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues. J Histochem Cytochem. 1984 Mar;32(3):289–298. doi: 10.1177/32.3.6198358. [DOI] [PubMed] [Google Scholar]
  34. Martinez-Hernandez A. The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver. Lab Invest. 1984 Jul;51(1):57–74. [PubMed] [Google Scholar]
  35. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  36. Modesti A., Kalebic T., Scarpa S., Togo S., Grotendorst G., Liotta L. A., Triche T. J. Type V collagen in human amnion is a 12 nm fibrillar component of the pericellular interstitium. Eur J Cell Biol. 1984 Nov;35(2):246–255. [PubMed] [Google Scholar]
  37. Monaghan P., Warburton M. J., Perusinghe N., Rudland P. S. Topographical arrangement of basement membrane proteins in lactating rat mammary gland: comparison of the distribution of type IV collagen, laminin, fibronectin, and Thy-1 at the ultrastructural level. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3344–3348. doi: 10.1073/pnas.80.11.3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moor H., Bellin G., Sandri C., Akert K. The influence of high pressure freezing on mammalian nerve tissue. Cell Tissue Res. 1980;209(2):201–216. doi: 10.1007/BF00237626. [DOI] [PubMed] [Google Scholar]
  39. Mynderse L. A., Hassell J. R., Kleinman H. K., Martin G. R., Martinez-Hernandez A. Loss of heparan sulfate proteoglycan from glomerular basement membrane of nephrotic rats. Lab Invest. 1983 Mar;48(3):292–302. [PubMed] [Google Scholar]
  40. Ott U., Odermatt E., Engel J., Furthmayr H., Timpl R. Protease resistance and conformation of laminin. Eur J Biochem. 1982 Mar;123(1):63–72. doi: 10.1111/j.1432-1033.1982.tb06499.x. [DOI] [PubMed] [Google Scholar]
  41. Paulsson M., Aumailley M., Deutzmann R., Timpl R., Beck K., Engel J. Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur J Biochem. 1987 Jul 1;166(1):11–19. doi: 10.1111/j.1432-1033.1987.tb13476.x. [DOI] [PubMed] [Google Scholar]
  42. Paulsson M., Dziadek M., Suchanek C., Huttner W. B., Timpl R. Nature of sulphated macromolecules in mouse Reichert's membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins. Biochem J. 1985 Nov 1;231(3):571–579. doi: 10.1042/bj2310571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Paulsson M., Fujiwara S., Dziadek M., Timpl R., Pejler G., Bäckström G., Lindahl U., Engel J. Structure and function of basement membrane proteoglycans. Ciba Found Symp. 1986;124:189–203. doi: 10.1002/9780470513385.ch11. [DOI] [PubMed] [Google Scholar]
  44. Paulsson M. Noncollagenous proteins of basement membranes. Coll Relat Res. 1987 Dec;7(6):443–461. doi: 10.1016/s0174-173x(87)80042-0. [DOI] [PubMed] [Google Scholar]
  45. Paulsson M., Yurchenco P. D., Ruben G. C., Engel J., Timpl R. Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane. J Mol Biol. 1987 Sep 20;197(2):297–313. doi: 10.1016/0022-2836(87)90125-2. [DOI] [PubMed] [Google Scholar]
  46. Pratt B. M., Madri J. A. Immunolocalization of type IV collagen and laminin in nonbasement membrane structures of murine corneal stroma. A light and electron microscopic study. Lab Invest. 1985 Jun;52(6):650–656. [PubMed] [Google Scholar]
  47. Rao C. N., Margulies I. M., Liotta L. A. Binding domain for laminin on type IV collagen. Biochem Biophys Res Commun. 1985 Apr 16;128(1):45–52. doi: 10.1016/0006-291x(85)91642-0. [DOI] [PubMed] [Google Scholar]
  48. Roth J. Light and electron microscopic localization of antigenic sites in tissue sections by the protein A-gold technique. Acta Histochem Suppl. 1984;29:9–22. [PubMed] [Google Scholar]
  49. Sanes J. R. Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol. 1982 May;93(2):442–451. doi: 10.1083/jcb.93.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sasaki M., Kato S., Kohno K., Martin G. R., Yamada Y. Sequence of the cDNA encoding the laminin B1 chain reveals a multidomain protein containing cysteine-rich repeats. Proc Natl Acad Sci U S A. 1987 Feb;84(4):935–939. doi: 10.1073/pnas.84.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sasaki M., Yamada Y. The laminin B2 chain has a multidomain structure homologous to the B1 chain. J Biol Chem. 1987 Dec 15;262(35):17111–17117. [PubMed] [Google Scholar]
  52. Schiff R., Rosenbluth J. Ultrastructural localization of laminin in rat sensory ganglia. J Histochem Cytochem. 1986 Dec;34(12):1691–1699. doi: 10.1177/34.12.3097120. [DOI] [PubMed] [Google Scholar]
  53. Semoff S., Hogan B. L., Hopkins C. R. Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy. EMBO J. 1982;1(10):1171–1175. doi: 10.1002/j.1460-2075.1982.tb00009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stephens H., Bendayan M., Gisiger V. Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction. Histochem J. 1985 Nov;17(11):1203–1220. doi: 10.1007/BF01002503. [DOI] [PubMed] [Google Scholar]
  55. Terranova V. P., Rao C. N., Kalebic T., Margulies I. M., Liotta L. A. Laminin receptor on human breast carcinoma cells. Proc Natl Acad Sci U S A. 1983 Jan;80(2):444–448. doi: 10.1073/pnas.80.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Timpl R. Antibodies to collagens and procollagens. Methods Enzymol. 1982;82(Pt A):472–498. doi: 10.1016/0076-6879(82)82079-x. [DOI] [PubMed] [Google Scholar]
  57. Timpl R., Dziadek M., Fujiwara S., Nowack H., Wick G. Nidogen: a new, self-aggregating basement membrane protein. Eur J Biochem. 1983 Dec 15;137(3):455–465. doi: 10.1111/j.1432-1033.1983.tb07849.x. [DOI] [PubMed] [Google Scholar]
  58. Timpl R., Dziadek M. Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol. 1986;29:1–112. [PubMed] [Google Scholar]
  59. Timpl R., Paulsson M., Dziadek M., Fujiwara S. Basement membranes. Methods Enzymol. 1987;145:363–391. doi: 10.1016/0076-6879(87)45021-0. [DOI] [PubMed] [Google Scholar]
  60. Timpl R., Wiedemann H., van Delden V., Furthmayr H., Kühn K. A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem. 1981 Nov;120(2):203–211. doi: 10.1111/j.1432-1033.1981.tb05690.x. [DOI] [PubMed] [Google Scholar]
  61. Tohyama K., Ide C. The localization of laminin and fibronectin on the Schwann cell basal lamina. Arch Histol Jpn. 1984 Nov;47(5):519–532. doi: 10.1679/aohc.47.519. [DOI] [PubMed] [Google Scholar]
  62. Yurchenco P. D., Ruben G. C. Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network. J Cell Biol. 1987 Dec;105(6 Pt 1):2559–2568. doi: 10.1083/jcb.105.6.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yurchenco P. D., Tsilibary E. C., Charonis A. S., Furthmayr H. Models for the self-assembly of basement membrane. J Histochem Cytochem. 1986 Jan;34(1):93–102. doi: 10.1177/34.1.3510247. [DOI] [PubMed] [Google Scholar]

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