December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Analysis of Caveolae/Raft Microdomains in Retinal Pigment Epithelium
Author Affiliations & Notes
  • K Nishiyama
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • M Miyagi
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • JW Lee
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • H Sakaguchi
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • ME Rayborn
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • KG Shadrach
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • JW Crabb
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • JG Hollyfield
    Cole Eye Institute The Cleveland Clinic Foundation Cleveland OH
  • Footnotes
    Commercial Relationships   K. Nishiyama, None; M. Miyagi, None; J.W. Lee, None; H. Sakaguchi, None; M.E. Rayborn, None; K.G. Shadrach, None; J.W. Crabb, None; J.G. Hollyfield, None. Grant Identification: Support: NIH and FFB
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2900. doi:
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      K Nishiyama, M Miyagi, JW Lee, H Sakaguchi, ME Rayborn, KG Shadrach, JW Crabb, JG Hollyfield; Analysis of Caveolae/Raft Microdomains in Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2900.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Caveolae/raft is a unique membrane microdomain, where a variety of signaling molecules are associated, and has been intensively investigated in non-ocular systems. However, information about caveolae/raft in ocular tissues, especially retinal pigment epithelium (RPE) is limited. Only one study reported that RPE does not have caveolae formation, while caveolin-1, one of the major constituent molecules of caveolae, is abundant in RPE. In this study we extend the analysis of caveolae/raft in RPE. Methods: Human RPE cells obtained at autopsy and immortalized culture cell lines (RPE-J and ARPE-19 cells) were used. Antibodies against caveolae/raft markers, including caveolins-1, -2 and -3 and flotillins-1 and -2, were commercially obtained. RNA and protein were extracted and used for RT-PCR, Western blotting, immunoprecipitation, sucrose gradient fractionation and/or mass spectrometry. RPE was also fixed appropriately and prepared for immunohistochemistry, electron microscopy (EM) and/or immuno-EM. Results: RT-PCR and Western blotting showed that caveolins-1 and -2 and flotillins-1 and -2 were expressed in RPE, while caveolin-3 was not. Immunohistochemistry in donor RPE demonstrated a basolateral distribution of caveolins and flotillins. EM showed that donor RPE cells possess occasional caveolae-like formations. Cell culture experiments revealed physical interaction between caveolins-1 and -2 and showed flotillin-1 content was elevated following treatment with retinoic acid. Sucrose gradient fractionation, in combination with Western blotting, showed that caveolins and flotillins were specifically restricted to the caveolae/raft fraction of cultured RPE cells. The caveolae/raft fraction of ARPE-19 cells was analyzed by mass-spectrometry, which identified several signaling molecules, including integrins and G-proteins. Immuno-EM study of caveolins and flotillins in RPE and further protein identification analysis of the molecules localizing to caveolae/raft microdomains in RPE are currently underway. Conclusion: The present data shows that RPE cells possess biochemical raft microdomains and histological caveolae-like structures. The data also show that caveolins and flotillins are specific markers for caveolae/raft fraction in the RPE, where caveolins interact with signaling molecules. Furthermore, elevation of flotillins-1 may be associated with RPE polarization.

Keywords: 567 retinal pigment epithelium • 342 cell membrane/membrane specializations • 526 protein purification and characterization 
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