July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Lens Aquaporin 0 (AQP0) interacts with negatively charged lipids in the fiber cell membrane to promote cell-to-cell adhesion
Author Affiliations & Notes
  • Kulandaiappan Varadaraj
    Physiology and Biophysics, State University of New York, Stony Brook, New York, United States
    SUNY Eye Institute, Syracuse, New York, United States
  • Sindhu S Kumari
    Physiology and Biophysics, State University of New York, Stony Brook, New York, United States
  • Footnotes
    Commercial Relationships   Kulandaiappan Varadaraj, None; Sindhu Kumari, None
  • Footnotes
    Support  NIH-NEI Grants EY020506 and EY026155
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3486. doi:
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      Kulandaiappan Varadaraj, Sindhu S Kumari; Lens Aquaporin 0 (AQP0) interacts with negatively charged lipids in the fiber cell membrane to promote cell-to-cell adhesion. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3486.

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

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Abstract

Purpose :
To find out the possible mechanism by which AQP0 exerts cell-to-cell adhesion between fiber cells, using newly-devised methods involving cell culture, lipid vesicles, and WT and AQP0 knockout (AQP0-KO) mouse lens fiber cell membrane (FCM) vesicles (FCMVs).

Methods : Adhesion-deficient normal fibroblast L-cells (sham) of mouse or those transfected with and expressing mouse intact AQP0 (263AA), C-terminal cleaved AQP0 (CTC-AQP0, 246AA) or human AQP1 were used. Large unilamellar vesicles (LUVs) of negatively charged L-α-phosphatidylserine (PS) and neutral phosphatidylcholine (PC) were prepared. WT and AQP0-KO mice (C57BL/6J strain) lens fiber cells from outer cortex, which contains intact AQP0, and inner cortex, which contains intact and CTC-AQP0, were isolated and FCMVs were prepared. Western blotting was done to verify the expression of AQP0 in cell lines and lenses, and adhesion assays were performed.

Results : Western blotting of proteins extracted from L-cells transfected with intact or CTC-AQP0 showed expression of ~28 or ~26 kDa proteins, respectively; FCM proteins of WT and AQP0-KO revealed the presence and absence of AQP0, respectively. L-cells expressing intact or CTC-AQP0 promoted adhesion significantly (P<0.001) compared to control cells or those expressing AQP1. Intact- or CTC-AQP0-expressing cells significantly (P<0.001) promoted adhesion to negatively charged PS lipid vesicles as opposed to neutral PC vesicles; these cells also promoted adhesion of mouse lens FCMVs of WT, and AQP0-KO significantly (P <0.001). However, when FCMVs of WT or AQP0-KO were plated over control L-cells, only WT vesicles significantly adhered. After incubating with extracellular domain-specific anti-AQP0 antibody, L-cells expressing intact or CTC-AQP0 showed significant reduction (P<0.001) in adhesion to FCMVs of AQP0-KO. WT FCMVs from outer cortex with intact AQP0, or inner cortex with intact and cleaved forms promoted adhesion to normal L-cells, without any statistically significant difference in adhesion efficiency (P>0.05).

Conclusions : Both intact and CTC-AQP0 expressed in L-cells promoted adhesion to AQP0-KO FMCVs as well as with negatively charged lipid membrane vesicles. These data suggest that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote fiber cell-to-cell adhesion.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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