April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
A free C-terminus is Essential for the Secretion of αB-crystallin via Exosomes in Human Adult Retinal Pigment Epithelial Cells in Culture
Author Affiliations & Notes
  • Rajendra K. Gangalum
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California
  • Zhe Jing
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California
  • Suraj P. Bhat
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California
    Molecular Biology Institute and Brain Research Institute,, University of California, Los Angeles, California
  • Footnotes
    Commercial Relationships  Rajendra K. Gangalum, None; Zhe Jing, None; Suraj P. Bhat, None
  • Footnotes
    Support  NIH Grant 1R01EY006044
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3339. doi:
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      Rajendra K. Gangalum, Zhe Jing, Suraj P. Bhat; A free C-terminus is Essential for the Secretion of αB-crystallin via Exosomes in Human Adult Retinal Pigment Epithelial Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3339.

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

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Abstract

Purpose: : αB-crystallin (αB) is a small heat shock protein that is known to protect cells in culture against stress. It is also a component of drusen associated with age-related macular degeneration (AMD). We have recently shown that this protein is secreted out of the human ARPE19 cells in culture via microvesicles (exosomes). These studies were undertaken to understand the molecular events associated with the secretion of αB from these cells.

Methods: : Two hybrid proteins were made: αB-GFP (with tGFP attached in-frame on the c-terminus of αB) and tGFP-αB (with tGFP attached in frame on the N-terminus of αB). Stable ARPE19 cell lines that express hybrid αB/tGFP (turbo Green Fluorescent Protein) proteins under the control of the homologous αB promoter were generated. Time-lapse Confocal microscopy was used to follow secretion of the recombinant molecules from ARPE cells. Sucrose density gradients, immunoblotting and Confocal fluorescent microscopy were used to follow the distribution of hybrid proteins in the cells.

Results: : Out of the two hybrid proteins only GFP-αB (GFP on the N-terminus of αB) is secreted out of the RPE cells. The other recombinant molecule αB-GFP is not found in the medium. Further analyses shows that GFP-αB is found in the exosomes; it can be followed by time-lapse Confocal imaging exiting the cell into the medium. The GFP-αB protein seems to localize (in the perinuclear Golgi) and distribute ‘normally’ in the RPE cells. On the other hand αB-GFP aggregates are seen in the cell at non-specific sites (as also assessed by sucrose density gradient fractionation) and are unable to exit the cell.

Conclusions: : These data indicate that a free C-terminus is important for the secretion of αB-crystallin out of ARPE cells via exosomes.

Keywords: crystallins • cell-cell communication • retinal pigment epithelium 
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