Advertisement

Journal of Neurocytology

, Volume 24, Issue 5, pp 371–388 | Cite as

Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembly

  • Joseph C. Besharse
  • Mary G. Wetzel
Article

Summary

Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors ofXenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5–6 loop regions of bovine opsin. This was done on isolated retinas incubatedin vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining ofXenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the ‘fracture-label’ technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.

Keywords

Outer Segment Basal Disc Immune Epitope Segment Disc Outer Segment Disc 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adamus, G., McDowell, J. H., Arendt, A., Hargrave, P. A., Smyk-Randall, E. &Sheehan, J. (1987) Structure, function and topography of rhodopsin as determined using monoclonal antibodies. InBiophysical Studies of Retinal Proteins, (edited byEbrey, T. G., Fraunfelder, H., Honig, H. &Nakanishi, K.) pp. 86–94. Urbana: University of Illinois Press.Google Scholar
  2. Adamus, G., Arendt, A., Zam, Z. S., McDowell, J. H. &Hargrave, P. A. (1988) Use of peptides to select for antirhodopsin antibodies with desired amino acid sequence specificities.Peptide Research 1, 42–7.Google Scholar
  3. Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. H. &Hargrave, P. A. (1991) Antirhodopsin monoclonal antibodies of defined specificity: characterization and application.Vision Research 31, 17–31.Google Scholar
  4. Besharse, J. C. (1980) Light and membrane biogenesis in rod photoreceptors of vertebrates. InThe Effects of Constant Light on Visual Processes (edited byWilliams, T. P. &Baker, B. N.) pp. 409–31. New York: Plenum Press.Google Scholar
  5. Besharse, J. C. (1986) Photosensitive membrane turnover: differential membrane domains and cell-cell interaction. InThe Retina. A Model for Cell Biological Studies. Part 1 (edited byAdler, R. &Farber, D.) pp. 297–352. New York: Academic Press.Google Scholar
  6. Beharse, J. C. &Dunis, D. A. (1983) Rod photoreceptor disc shedding in eye cups: relationship to bicarbonate and amino acids.Experimental Eye Research 36, 567–80.Google Scholar
  7. Besharse, J. C. &Horsat, C. J. (1990) The photoreceptor connecting cilium: a model for the transition zone. InCiliary and Flagellar Membanes (edited byBloodgood, R. A.) pp. 389–417. New York: Plenum Press.Google Scholar
  8. Besharse, J. C. &Pfenninger, K. H. (1980) Membrane assembly in retinal photoreceptors. I. Freeze fracture analysis of cytoplasmic vesicles in relationship to disc assembly.Journal of Cell Biology 87, 451–63.Google Scholar
  9. Besharse, J. C., Hollyfield, J. G. &Rayborn, M. E. (1977a) Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light.Journal of Cell Biology 75, 507–27.Google Scholar
  10. Besharse, J. C., Hollyfield, J. G. &Rayborn, M. E. (1977b) Photoreceptor outer segments: accelerated membrane renewal in rods after exposure to light.Science 196, 536–8.Google Scholar
  11. Besharse, J. C., Forestner, D. M. &Defoe, D. M. (1985) Membrane assembly in retinal photoreceptors. III. Distinct membrane domains of the connecting cilium of developing rods.Journal of Neuroscience 5, 1035–48.Google Scholar
  12. Biernbaum, M. S. &Bownds, M. D. (1985) Frog rod outer segments with attached inner segment ellipsoids as anin vitro model for photoreceptors on the retina.Journal of General Physiology 85, 83–105.Google Scholar
  13. Blanks, J. C., Müllen, R. J. &Lavail, M. M. (1982) Retinal degeneration in the pcd cerebellar mutant mouse. II. Electron microscopic analysis.Journal of Comparative Neurology 212, 231–46.Google Scholar
  14. Bok, D. (1985) Retinal photoreceptor-pigment epithelium interactions.Investigative Ophthalmology and Visual Science 26, 1659–94.Google Scholar
  15. Chaitin, M. H. (1992) Double immunogold localization of opsin and actin in the cilium of developing mouse photoreceptors.Experimental Eye Research 54, 261–7.Google Scholar
  16. Compans, R. W. &Roberts, P. C. (1994) Viruses as model systems in cell biology.Methods in Cell Biology,43, 3–42.Google Scholar
  17. Corless, J. M., Cobbs, W. H., III, Costello, M. J. &Robertson, J. D. (1976) On the asymmetry of frog retinal rod outer segment disc membranes.Experimental Eye Research 23, 295–324.Google Scholar
  18. Defoe, D. M. &Besharse, J. C. (1985) Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies.Journal of Neurosciences 5, 1023–34.Google Scholar
  19. Deretic, D. &Papermaster, D. S. (1991) Polarized sorting of rhodopsin on post-Goldi membranes in frog retinal photoreceptor cells.Journal of Cell Biology 113, 1281–93.Google Scholar
  20. Fliesler, S. J., Rayborn, M. E. &Hollyfield, J. G. (1985) Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.Journal of Cell Biology 100, 574-S7.Google Scholar
  21. Hargrave, P. A. (1982) Rhodopsin chemistry, structure and topography. InProgress in Retinal Research, Vol. 1 (edited byOsborne, N. &Chader, G.) pp. 1–51. New York: Pergamon Press.Google Scholar
  22. Hasty, D. L. &Hay, E. D. (1978) Freeze-fracture studies of the developing cell surface. II. Particle-free membrane blisters on glutaraldehyde-fixed corneal fibroblasts are artefacts.Journal of Cell Biology 78, 756–68.Google Scholar
  23. Hicks, D. &Barnstable, C. J. (1986) Lectin and antibody labelling of developing rat photoreceptor cells: an electron microscopic immunocytochemical study.Journal of Neurocytology 15, 219–30.Google Scholar
  24. Hicks, D. &Barnstable, C. J. (1987) Different rhodopsin monoclonal antibodies reveal different binding patterns on developing and adult rat retinas.Journal of Histochemistry and Cytochemistry 35, 1317–28.Google Scholar
  25. Hicks, D. &Molday, R. S. (1986) Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin.Experimental Eye Research 42, 55–71.Google Scholar
  26. Hollyfield, J. G. &Rayborn, M. E. (1979) Photoreceptor outer segment development: light and dark regulate the rate of membrane addition and loss.Investigative Ophthalmology and Visual Science 18, 117–32.Google Scholar
  27. Hollyfield, J. G., Rayborn, M. E., Verner, G. E., Maude, M. B. &Anderson, R. E. (1982) Membrane addition to rod photoreceptor outer segments: light stimulates membrane assembly in the absence of increased membrane biosynthesis.Investigative Ophthalmology and Visual Sciences 22, 417–27.Google Scholar
  28. Horst, C. J., Forestner, D. M. &Besharse, J. C. (1987) Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.Journal of Cell Biology 105, 2973–87.Google Scholar
  29. Horst, C. J., Johnson, L. V. &Besharse, J. C. (1990) Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunological epitope.Cell Motility and the Cytoskeleton 17, 329–4.Google Scholar
  30. Locket, N. A. (1973) Possible discontinuous retinal rod outer segment formation inLatimeria chalumnae.Nature 244, 308–9.Google Scholar
  31. Matsusaka, T. (1974) Membrane particles of the connecting cilium.Journal of Ultrastructure Research 48, 305–12.Google Scholar
  32. Miyaguchi, K. &Hashimoto, P. H. (1992) Evidence for the transport of opsin in the connecting cilium and basal rod outer segment in rat retina: rapid-freeze, deep-etch and horseradish peroxidase labelling studies.Journal of Neurocytology 21, 449–57.Google Scholar
  33. Molday, R. S. (1988) Monoclonal antibodies to rhodopsin and other proteins of rod outer segments.Progress in Retinal Research 8, 173–209.Google Scholar
  34. Molday, R. S. &Mackenzie, D. (1983) Monoclonal antibodies to rhodopsin: characterization, cross reactivity and application as structural probes.Biochemistry 22, 653–60.Google Scholar
  35. Muresan, V. &Besharse, J. C. (1994) Complex intermolecular interactions maintain a stable linkage between the photoreceptor connecting cilium axoneme and plasma membrane.Cell Motility and the Cytoskeleton 28, 213–30.Google Scholar
  36. Nir, I. &Papermaster, D. S. (1983) Differential distribution of opsin in the plasma membrane of frog photo-receptors: an immunocytochemical study.Investigative Ophthalmology and Visual Science 24, 868–78.Google Scholar
  37. Nir, I. &Papermaster, D. S. (1986) Immunocytochemical localization of opsin in the inner segment and ciliary plasma membrane of photoreceptors in retinas of rds mutant mice.Investigative Ophthalmology and Visual Science 27, 836–40.Google Scholar
  38. Nir, I., Cohen, D. &Papermaster, D. S. (1984) Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors.Journal of Cell Biology 98, 1788–95.Google Scholar
  39. Nir, I., Cohen, D. &Papermaster, D. S. (1987) Fused inner-outer segments in developing mouse photoreceptors.Investigative Ophthalmology and Visual Science 28, 287 (Abstract).Google Scholar
  40. Papermaster, D. S. &Schneider, B. G. (1982) Biosynthesis and morphogenesis of outer segment membranes in vertebrate photoreceptor cells. InCell Biology of the Eye (edited byMcDevitt, D.) pp. 475–531. New York: Academic Press.Google Scholar
  41. Papermaster, D. S., Converse, C. A. &Siu, J. (1975) Membrane biosynthesis in the frog retina: opsin transport in the photoreceptor cell.Biochemistry 14, 2438–42.Google Scholar
  42. Papermaster, D. S., Schneider, B. G., Zohn, M. A. &Kraehenbuhl, J. P. (1978) Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells: an electron microscopic analysis of cross-linked albumin-embedded retina.Journal of Cell Biology 77, 196–210.Google Scholar
  43. Papermaster, D. S., Schneider, B. G. &Besharse, J. C. (1985) Vesicular transport of newly synthesized opsin from the Golgi apparatus towards the rod outer segment.Investigative Ophthalmology and Visual Sciences 26, 1386–404.Google Scholar
  44. Peters, K. R., Palade, G. E., Schneider, B. S. &Papermaster, D. S. (1983) Fine structure of a periciliary ridge complex of frog retinal rod cells revealed by ultrahigh resolution scanning electron microscopy.Journal of Cell Biology 96, 265–76.Google Scholar
  45. Pinto Da Silva, P., Parkinson, C. &Dwyer, N. (1981) Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fracture faces.Journal of Histochemistry and Cytochemistry 29, 917–28.Google Scholar
  46. Polans, A. S., Altman, L. G. &Papermaster, D. S. (1986) Immunocytochemical binding of anti-opsin N-terminal specific antibodies to the extracellular surface of rod outer segment plasma membranes: fixation induces antibody binding.Journal of Histochemistry and Cytochemistry 34, 659–64.Google Scholar
  47. Richardson, T. M. (1969) Cytoplasmic and ciliary connections between the inner and outer segments of mammalian visual receptors.Vision Research 9, 727–31.Google Scholar
  48. Róhlich, P. (1975) The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia.Cell and Tissue Research 166, 421–30.Google Scholar
  49. Róhlich, P., Adamus, G., McDowell, J. H. &Hargrave, P. A. (1989a) Binding pattern of antirhodopsin monoclonal antibodies to photoreceptor cells: an immunocytochemical study.Experimental Eye Research 49, 999–1013.Google Scholar
  50. Róhlich, P., Szél, A. &Papermaster, D. S. (1989b) Immunocytochemical reactivity ofXenopus laevis retinal rods and cones with several monoclonal antibodies to visual pigments.Journal of Comparative Neurology 290, 105–17.Google Scholar
  51. Saha, M. S. &Grainger, R. M. (1993) Early opsin expression inXenopus embryos precedes photoreceptor differentiation.Molecular Brain Research 17, 307–18.Google Scholar
  52. Sale, W. S., Besharse, J. C. &Piperno, G. (1988) Distribution of acetylated α-tubulin in retina andin vitro- assembled microtubules.Cell Motility and the Cytoskeleton 9, 243–53.Google Scholar
  53. Spencer., M., Detwiler, P. B. &Bunt-Milam, A. H. (1988) Distribution of membrane proteins in mechanically dissociated retinal rods.Investigative Ophthalmology and Visual Sciences 29, 1012–20.Google Scholar
  54. Szél, A. &Rölich, P. (1985) Localization of visual pigment antigens to photoreceptor cells with different oil droplets in chicken retina.Acta Biologica Hungarica 36, 319–24.Google Scholar
  55. Szél, A., Takacs, L., Monstori, E., Diamantstein, T., Vigh-Teichman, I. &Rohlich, P. (1986) Monoclonal antibody recognizing cone visual pigment.Experimental Eye Research 43, 871–83.Google Scholar
  56. Tokuyasu, K. T. (1989) Use of poly (vinyl pyrrolidone) and poly (vinyl alcohol) for cryoultramicrotomy.Histochemical Journal 21, 163–71.Google Scholar
  57. Townes-Anderson, E. (1989) Intersegmental fusion in rod photoreceptors of the salamander retina.Investigative Ophthalmology and Visual Sciences.30, 156 (Abstract Supplement).Google Scholar
  58. Townes-Anderson, E., Dacheux, R. F. &Raviola, E. (1988) Rod photoreceptors dissociated from the adult rabbit retina.The Journal of Neuroscience 8, 320–31.Google Scholar
  59. Usukura, J. &Bok, D. (1987) Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay.Experimental Eye Research 45, 501–15.Google Scholar
  60. Wetzel, M. G., Bendala-Tufanisco, E. &Besharse, J. C. (1993) Tunicamycin does not inhibit transport of phosphatidylinositol toXenopus rod outer segments.Journal of Neurocytology 22, 397–412.Google Scholar
  61. Young, R. W. (1967) The renewal of photoreceptor cell outer segments.Journal of Cell Biology 83, 61–72.Google Scholar
  62. Young, R. W. (1968) Passage of newly formed protein through the connecting cilium of retinal rods in the frog.Journal of Ultrastructure Research 23, 462–73.Google Scholar

Copyright information

© Chapman and Hall 1995

Authors and Affiliations

  • Joseph C. Besharse
    • 1
  • Mary G. Wetzel
    • 1
  1. 1.Department of Anatomy and Cell BiologyThe University of Kansas Medical CenterKansas CityUSA

Personalised recommendations