Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Sep 1;97(3):749–755. doi: 10.1083/jcb.97.3.749

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

PMCID: PMC2112550  PMID: 6411740

Abstract

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Full Text

The Full Text of this article is available as a PDF (4.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alroy J., Merk F. B., Goyal V., Ucci A. Heterogeneous distribution of filipin-sterol complexes in nuclear membranes. Biochim Biophys Acta. 1981 Dec 7;649(2):239–243. doi: 10.1016/0005-2736(81)90411-9. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. E. Lipids of ocular tissues. IV. A comparison of the phospholipids from the retina of six mammalian species. Exp Eye Res. 1970 Oct;10(2):339–344. doi: 10.1016/s0014-4835(70)80046-x. [DOI] [PubMed] [Google Scholar]
  3. Andrews L. D., Cohen A. I. Freeze-fracture evidence for the presence of cholesterol in particle-free patches of basal disks and the plasma membrane of retinal rod outer segments of mice and frogs. J Cell Biol. 1979 Apr;81(1):215–228. doi: 10.1083/jcb.81.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen Y. S., Hubbell W. L. Temperature- and light-dependent structural changes in rhodopsin-lipid membranes. Exp Eye Res. 1973 Dec 24;17(6):517–532. doi: 10.1016/0014-4835(73)90082-1. [DOI] [PubMed] [Google Scholar]
  5. Cherry R. J., Müller U., Holenstein C., Heyn M. P. Lateral segregation of proteins induced by cholesterol in bacteriorhodopsin-phospholipid vesicles. Biochim Biophys Acta. 1980 Feb 15;596(1):145–151. doi: 10.1016/0005-2736(80)90179-0. [DOI] [PubMed] [Google Scholar]
  6. Corless J. M., Cobbs W. H., 3rd, Costello M. J., Robertson J. D. On the asymmetry of frog retinal rod outer segment disk membranes. Exp Eye Res. 1976 Sep;23(3):295–324. doi: 10.1016/0014-4835(76)90130-5. [DOI] [PubMed] [Google Scholar]
  7. Elias P. M., Friend D. S., Goerke J. Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin. J Histochem Cytochem. 1979 Sep;27(9):1247–1260. doi: 10.1177/27.9.479568. [DOI] [PubMed] [Google Scholar]
  8. FINE B. S., ZIMMERMAN L. E. OBSERVATIONS ON THE ROD AND CONE LAYER OF THE HUMAN RETINA. A LIGHT AND ELECTRON MICROSCOPIC STUDY. Invest Ophthalmol. 1963 Oct;2:446–459. [PubMed] [Google Scholar]
  9. Fain G. L., Quandt F. N., Gerschenfeld H. M. Calcium-dependent regenerative responses in rods. Nature. 1977 Oct 20;269(5630):707–710. doi: 10.1038/269707a0. [DOI] [PubMed] [Google Scholar]
  10. Feltkamp C. A., van der Waerden A. W. Low temperature-induced displacement of cholesterol and intramembrane particles in nuclear membranes of mouse leukemia cells. Cell Biol Int Rep. 1982 Feb;6(2):137–145. doi: 10.1016/0309-1651(82)90090-x. [DOI] [PubMed] [Google Scholar]
  11. Friend D. S. Freeze-fracture alterations in guinea pig sperm membranes preceding gamete fusion. Soc Gen Physiol Ser. 1980;34:153–165. [PubMed] [Google Scholar]
  12. Fujita H., Ishimura K., Matsuda H. Freeze-fracture images on filipin-sterol complexes in the thyroid follicle epithelial cell of mice with special regard to absence of cholesterol at the site of micropinocytosis. Histochemistry. 1981;73(1):57–63. doi: 10.1007/BF00493133. [DOI] [PubMed] [Google Scholar]
  13. Garcia-Segura L. M., Baetens D., Orci L. Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons. Brain Res. 1982 Feb 25;234(2):494–499. doi: 10.1016/0006-8993(82)90893-9. [DOI] [PubMed] [Google Scholar]
  14. Montesano R. Inhomogeneous distribution of filipin-sterol complexes in smooth muscle cell plasma membrane. Nature. 1979 Jul 26;280(5720):328–329. doi: 10.1038/280328a0. [DOI] [PubMed] [Google Scholar]
  15. Montesano R. Inhomogeneous distribution of filipin-sterol complexes in the ciliary membrane of rat tracheal epithelium. Am J Anat. 1979 Sep;156(1):139–145. doi: 10.1002/aja.1001560115. [DOI] [PubMed] [Google Scholar]
  16. Montesano R., Vassalli P., Perrelet A., Orci L. Distribution of filipin-cholesterol complexes at sites of exocytosis - a freeze-fracture study of degranulating mast cells. Cell Biol Int Rep. 1980 Nov;4(11):975–984. doi: 10.1016/0309-1651(80)90170-8. [DOI] [PubMed] [Google Scholar]
  17. Nakajima Y., Bridgman P. C. Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions. J Cell Biol. 1981 Feb;88(2):453–458. doi: 10.1083/jcb.88.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohki K., Nozawa Y., Ohnishi S. I. Interaction of polyene antibiotics with sterols in phosphatidylcholine bilayer membranes as studied by spin probes. Biochim Biophys Acta. 1979 Jun 13;554(1):39–50. doi: 10.1016/0005-2736(79)90004-x. [DOI] [PubMed] [Google Scholar]
  19. Peters K. R., Palade G. E., Schneider B. G., Papermaster D. S. Fine structure of a periciliary ridge complex of frog retinal rod cells revealed by ultrahigh resolution scanning electron microscopy. J Cell Biol. 1983 Jan;96(1):265–276. doi: 10.1083/jcb.96.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Robenek H., Melkonian M. Sterol-deficient domains correlate with intramembrane particle arrays in the plasma membrane of Chlamydomonas reinhardii. Eur J Cell Biol. 1981 Oct;25(2):258–264. [PubMed] [Google Scholar]
  21. Robinson J. M., Karnovsky M. J. Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem. 1980 Feb;28(2):161–168. doi: 10.1177/28.2.6766487. [DOI] [PubMed] [Google Scholar]
  22. Robinson J. M., Karnovsky M. J. Specializations in filopodial membranes at points of attachment to the substrate. J Cell Biol. 1980 Dec;87(3 Pt 1):562–568. doi: 10.1083/jcb.87.3.562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sekiya T., Kitajima Y., Nozawa Y. Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy. Biochim Biophys Acta. 1979 Jan 19;550(2):269–278. doi: 10.1016/0005-2736(79)90213-x. [DOI] [PubMed] [Google Scholar]
  24. Severs N. J. Localization of cholesterol in the Golgi apparatus of cardiac muscle cells. Experientia. 1981 Nov 15;37(11):1195–1198. doi: 10.1007/BF01989915. [DOI] [PubMed] [Google Scholar]
  25. Severs N. J. Plasma membrane cholesterol in myocardial muscle and capillary endothelial cells. Distribution of filipin-induced deformations in freeze-fracture. Eur J Cell Biol. 1981 Oct;25(2):289–299. [PubMed] [Google Scholar]
  26. Sommer J. R., Dolber P. C., Taylor I. Filipin-cholesterol complexes in the sarcoplasmic reticulum of frog skeletal muscle. J Ultrastruct Res. 1980 Sep;72(3):272–285. doi: 10.1016/s0022-5320(80)90064-7. [DOI] [PubMed] [Google Scholar]
  27. Tillack T. W., Kinsky S. C. A freeze-etch study of the effects of filipin on liposomes and human erythrocyte membranes. Biochim Biophys Acta. 1973 Sep 27;323(1):43–54. doi: 10.1016/0005-2736(73)90430-6. [DOI] [PubMed] [Google Scholar]
  28. Verkleij A. J., de Kruijff B., Gerritsen W. F., Demel R. A., van Deenen L. L., Ververgaert P. H. Freeze-etch electron microscopy of erythrocytes, Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B. Biochim Biophys Acta. 1973 Jan 26;291(2):577–581. doi: 10.1016/0005-2736(73)90509-9. [DOI] [PubMed] [Google Scholar]
  29. Young R. W., Droz B. The renewal of protein in retinal rods and cones. J Cell Biol. 1968 Oct;39(1):169–184. doi: 10.1083/jcb.39.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES