September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Usher proteins are engaged in vesicular trafficking in human retinal pigment epithelial and fibroblasts cells
Author Affiliations & Notes
  • Bhagwat V. Alapure
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Centre, New Orleans, Louisiana, United States
  • Marianne Hathaway
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Centre, New Orleans, Louisiana, United States
  • Jennifer J Lentz
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Centre, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Bhagwat Alapure, None; Marianne Hathaway, None; Jennifer Lentz, None
  • Footnotes
    Support  The National Institutes of Health, Foundation Fighting Blindness, Eye on Jacob Foundation, Usher in Sight and Sound, and Usher 2020 Foundation.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 255. doi:
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      Bhagwat V. Alapure, Marianne Hathaway, Jennifer J Lentz; Usher proteins are engaged in vesicular trafficking in human retinal pigment epithelial and fibroblasts cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):255.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Usher syndrome (Usher) is the most common genetic condition that affects both hearing and vision. Sixteen genes are associated with 3 clinical subtypes. Among the Usher genes, thirteen are known and studies on their encoded proteins suggest that they interact together and function in multiprotein complexes. Individual Usher proteins, including myosin VIIa (USH1B), harmonin (USH1C), cadherin 23 (USH1D), protocadherin 15 (USH1F), SANS (USH1G), CIB2 (USH1J), usherin, (USH2A), VLGR1/GPR98 (USH2C), whirlin (USH2D), clarin-1 (USH3A), HARS (USH3B), PZDZ7 (USH modifier) and CEP250 (atypical USH), are expressed in hair cells and photoreceptors and are predicted to perform a wide range of functions including intracellular trafficking, scaffolding, cell adhesion and signaling. The exact function of these proteins and mechanism by which they interact is unclear. The purpose of this study is to determine the sub-cellular localization of Usher proteins and the effect of blocking trafficking on their expression in human retinal pigment epithelial (ARPE-19) and human dermal fibroblast (hFB) cultured cells.

Methods : ARPE-19 and hFB cells were cultured with and without trafficking inhibitors Brefeldin A (BFA) and Membrane Traffic Inhibitor A5. The expression and sub-cellular localization of Usher proteins were investigated using immunohistochemistry techniques. Immunofluorescence intensity was quantified and analyzed using ImageJ Software.

Results : All Usher proteins were found expressed in both ARPE-19 and hFB cells. All were found in the cytoplasm associated with the endoplasmic reticulum. Myosin VIIa, harmonin and cadherin 23 were also observed co-localized with the Golgi apparatus. Additionally, myosin VIIa, harmonin, cadherin 23, protocadherin 15, SANS, usherin and VLGR1 were found associated with F-actin showing a punctate pattern; whereas SANS appeared to co-localize along the long F-actin fibers. Lysosomal co-localization was not observed with any of the Usher proteins. Reduced expression of all Usher proteins was observed in the presence of BFA and A5 inhibitors.

Conclusions : These data suggest Usher proteins are involved in intracellular trafficking between the ER and Golgi in ARPE-19 and hFB cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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