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. 1987 Dec 1;105(6):2973–2987. doi: 10.1083/jcb.105.6.2973

Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium

PMCID: PMC2114726  PMID: 3693403

Abstract

The ciliary base is marked by a transition zone in which Y-shaped cross- linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y- shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA- binding component seen in cytochemical experiments.

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Selected References

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  1. Anderson R. G. Isolation of ciliated or unciliated basal bodies from the rabbit oviduct. J Cell Biol. 1974 Feb;60(2):393–404. doi: 10.1083/jcb.60.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. G. The three-dimensional structure of the basal body from the rhesus monkey oviduct. J Cell Biol. 1972 Aug;54(2):246–265. doi: 10.1083/jcb.54.2.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bardele C. F. Mapping of highly ordered membrane domains in the plasma membrane of the ciliate Cyclidium glaucoma. J Cell Sci. 1983 May;61:1–30. doi: 10.1242/jcs.61.1.1. [DOI] [PubMed] [Google Scholar]
  4. Besharse J. C., Forestner D. M., Defoe D. M. Membrane assembly in retinal photoreceptors. III. Distinct membrane domains of the connecting cilium of developing rods. J Neurosci. 1985 Apr;5(4):1035–1048. doi: 10.1523/JNEUROSCI.05-04-01035.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Besharse J. C., Pfenninger K. H. Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly. J Cell Biol. 1980 Nov;87(2 Pt 1):451–463. doi: 10.1083/jcb.87.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhavanandan V. P., Katlic A. W. The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. J Biol Chem. 1979 May 25;254(10):4000–4008. [PubMed] [Google Scholar]
  7. Bloodgood R. A. Motility occurring in association with the surface of the Chlamydomonas flagellum. J Cell Biol. 1977 Dec;75(3):983–989. doi: 10.1083/jcb.75.3.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carey D. J., Todd M. S. A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells. J Biol Chem. 1986 Jun 5;261(16):7518–7525. [PubMed] [Google Scholar]
  9. Defoe D. M., Besharse J. C. Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies. J Neurosci. 1985 Apr;5(4):1023–1034. doi: 10.1523/JNEUROSCI.05-04-01023.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dentler W. L., Pratt M. M., Stephens R. E. Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase. J Cell Biol. 1980 Feb;84(2):381–403. doi: 10.1083/jcb.84.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunlap K. Localization of calcium channels in Paramecium caudatum. J Physiol. 1977 Sep;271(1):119–133. doi: 10.1113/jphysiol.1977.sp011993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fatt P. Proteins of vertebrate rod outer segments: a possible role for multiple forms of rhodopsin. Exp Eye Res. 1981 Jul;33(1):31–46. doi: 10.1016/s0014-4835(81)80079-6. [DOI] [PubMed] [Google Scholar]
  13. Fleischman D., Denisevich M., Raveed D., Pannbacker R. G. Association of guanylate cyclase with the axoneme of retinal rods. Biochim Biophys Acta. 1980 Jun 19;630(2):176–186. doi: 10.1016/0304-4165(80)90419-5. [DOI] [PubMed] [Google Scholar]
  14. Flower N. E. Particles within membranes: a freeze-etch view. J Cell Sci. 1971 Sep;9(2):435–441. doi: 10.1242/jcs.9.2.435. [DOI] [PubMed] [Google Scholar]
  15. GIBBONS I. R., GRIMSTONE A. V. On flagellar structure in certain flagellates. J Biophys Biochem Cytol. 1960 Jul;7:697–716. doi: 10.1083/jcb.7.4.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gilbert S. P., Sloboda R. D. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules. J Cell Biol. 1986 Sep;103(3):947–956. doi: 10.1083/jcb.103.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gilula N. B., Satir P. The ciliary necklace. A ciliary membrane specialization. J Cell Biol. 1972 May;53(2):494–509. doi: 10.1083/jcb.53.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hard R., Rieder C. L. Muciliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models. Tissue Cell. 1983;15(2):227–243. doi: 10.1016/0040-8166(83)90019-8. [DOI] [PubMed] [Google Scholar]
  19. Hennessey T. M., Andrews D., Nelson D. L. Biochemical studies of the excitable membrane of Paramecium tetraurelia. VII. Sterols and other neutral lipids of cells and cilia. J Lipid Res. 1983 May;24(5):575–587. [PubMed] [Google Scholar]
  20. Hicks D., Barnstable C. J. Lectin and antibody labelling of developing rat photoreceptor cells: an electron microscope immunocytochemical study. J Neurocytol. 1986 Apr;15(2):219–230. doi: 10.1007/BF01611658. [DOI] [PubMed] [Google Scholar]
  21. Hicks D., Molday R. S. Localization of lectin receptors on bovine photoreceptor cells using dextran-gold markers. Invest Ophthalmol Vis Sci. 1985 Jul;26(7):1002–1013. [PubMed] [Google Scholar]
  22. Johnson K. A. Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin. Annu Rev Biophys Biophys Chem. 1985;14:161–188. doi: 10.1146/annurev.bb.14.060185.001113. [DOI] [PubMed] [Google Scholar]
  23. King S. M., Otter T., Witman G. B. Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4717–4721. doi: 10.1073/pnas.82.14.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kjellén L., Pettersson I., Hök M. Cell-surface heparan sulfate: an intercalated membrane proteoglycan. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5371–5375. doi: 10.1073/pnas.78.9.5371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Machemer H., Ogura A. Ionic conductances of membranes in ciliated and deciliated Paramecium. J Physiol. 1979 Nov;296:49–60. doi: 10.1113/jphysiol.1979.sp012990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Monsigny M., Roche A. C., Sene C., Maget-Dana R., Delmotte F. Sugar-lectin interactions: how does wheat-germ agglutinin bind sialoglycoconjugates? Eur J Biochem. 1980 Feb;104(1):147–153. doi: 10.1111/j.1432-1033.1980.tb04410.x. [DOI] [PubMed] [Google Scholar]
  28. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  29. Nir I., Cohen D., Papermaster D. S. Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors. J Cell Biol. 1984 May;98(5):1788–1795. doi: 10.1083/jcb.98.5.1788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ogura A., Takahashi K. Artificial deciliation causes loss of calcium-dependent responses in Paramecium. Nature. 1976 Nov 11;264(5582):170–172. doi: 10.1038/264170a0. [DOI] [PubMed] [Google Scholar]
  31. Papermaster D. S., Schneider B. G., Besharse J. C. Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas. Invest Ophthalmol Vis Sci. 1985 Oct;26(10):1386–1404. [PubMed] [Google Scholar]
  32. Papermaster D. S., Schneider B. G., DeFoe D., Besharse J. C. Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors. J Histochem Cytochem. 1986 Jan;34(1):5–16. doi: 10.1177/34.1.2934469. [DOI] [PubMed] [Google Scholar]
  33. Piperno G., Fuller M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985 Dec;101(6):2085–2094. doi: 10.1083/jcb.101.6.2085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piperno G., LeDizet M., Chang X. J. Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol. 1987 Feb;104(2):289–302. doi: 10.1083/jcb.104.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Poo M., Cone R. A. Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature. 1974 Feb 15;247(5441):438–441. doi: 10.1038/247438a0. [DOI] [PubMed] [Google Scholar]
  36. Pratt M. M. Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity. J Cell Biol. 1986 Sep;103(3):957–968. doi: 10.1083/jcb.103.3.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rapraeger A. C., Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. A putative membrane proteoglycan associates quantitatively with lipid vesicles. J Biol Chem. 1983 Mar 25;258(6):3632–3636. [PubMed] [Google Scholar]
  38. Röhlich P. The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia. Cell Tissue Res. 1975 Aug 25;161(3):421–430. doi: 10.1007/BF00220009. [DOI] [PubMed] [Google Scholar]
  39. Schultz J. E., Klumpp S. Calcium/calmodulin-regulated guanylate cyclases in the ciliary membranes from Paramecium and tetrahymena. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:275–283. [PubMed] [Google Scholar]
  40. Suprenant K. A., Dentler W. L. Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins. J Cell Biol. 1982 Apr;93(1):164–174. doi: 10.1083/jcb.93.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Toda N., Doi A., Jimbo A., Matsumoto I., Seno N. Interaction of sulfated glycosaminoglycans with lectins. J Biol Chem. 1981 Jun 10;256(11):5345–5349. [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wood J. G., Sarinana F. O. The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase. Anal Biochem. 1975 Nov;69(1):320–322. doi: 10.1016/0003-2697(75)90597-7. [DOI] [PubMed] [Google Scholar]
  45. Woods A., Hök M., Kjellén L., Smith C. G., Rees D. A. Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts. J Cell Biol. 1984 Nov;99(5):1743–1753. doi: 10.1083/jcb.99.5.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Young R. W. The renewal of photoreceptor cell outer segments. J Cell Biol. 1967 Apr;33(1):61–72. doi: 10.1083/jcb.33.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]

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