April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Regulation of Retinoschisin Secretion by Actin Filaments
Author Affiliations & Notes
  • E. Kitamura
    Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California
  • D. B. Farber
    Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California
  • Footnotes
    Commercial Relationships  E. Kitamura, None; D.B. Farber, None.
  • Footnotes
    Support  NIH Grant RO1EY8285
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4156. doi:
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      E. Kitamura, D. B. Farber; Regulation of Retinoschisin Secretion by Actin Filaments. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4156.

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

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Purpose: : The XLRS1 gene encodes the protein retinoschisin that is distributed throughout the retina. Its amino acid sequence shows a secretory leader sequence, suggesting that retinoschisin is a secreted protein. We previously demonstrated that retinoschisin is mainly synthesized and secreted by photoreceptor cells in the outer retina. The purpose of this study was to investigate whether SNAREs are involved in retinoschisin secretion and whether this secretion is regulated by actin filaments.

Methods: : Co-localization of retinoschisin with SNAREs or with actin filaments was determined by double immunofluorescence staining of WERI-Rb1 retinoblastoma cells. Double-labeled fluorescent samples were observed under confocal microscopy. To assess the effects of cytoskeletal drugs on retinoschisin secretion, WERI-Rb1 cells were cultured in RPMI1640 media supplemented with various concentrations of actin-modifying drugs for 72 hours. Conditioned media and whole cell lysates were immunoblotted using the retinoschisin antibody and the intensity of the signals was measured with an AlphaImager. In order to analyze the actin structure of cells treated with cytoskeletal drugs, actin filaments were visualized by fluorescence microscopy after incubation with phalloidin-Alexa488.

Results: : Double immunolabeling revealed the partial co-localization of retinoschisin and SNAREs in WERI-Rb1 cells. In addition, retinoschisin co-localized with actin filaments in some areas of the cytoplasm and cell membrane. Depolymerization of actin filaments by cytochalasin D resulted in accumulation of puncta within cells. Retinoschisin secretion was drastically reduced by exposure to 2 uM cytochalasin D or 1 uM of the F-actin stabilizer, Jasplakinolide.

Conclusions: : Co-localization of retinoschisin with SNAREs suggested a potential role of SNAREs in exocytotic secretion of retinoschisin. Secretion analysis using cytoskeletal drugs showed that alteration of actin filaments has inhibitory effects on retinoschisin secretion, suggesting that actin filaments play an active role in regulating this process.

Keywords: cytoskeleton • drug toxicity/drug effects • microscopy: light/fluorescence/immunohistochemistry 

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