September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Endosome-dependent Rhodopsin trafficking for outer segment renewal
Author Affiliations & Notes
  • Ya-Chu Hsu
    Ophthalmology, Weill Cornell Medical College of Cornell University, New York, New York, United States
  • Jen-Zen Chuang
    Ophthalmology, Weill Cornell Medical College of Cornell University, New York, New York, United States
  • Roland Thuenauer
    Albert-Ludwigs University, Freiburg, Germany
  • Ching-Kang Jason Chen
    Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Yu-Ting Yan
    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
  • Ching-Hwa Sung
    Ophthalmology, Weill Cornell Medical College of Cornell University, New York, New York, United States
  • Footnotes
    Commercial Relationships   Ya-Chu Hsu, None; Jen-Zen Chuang, None; Roland Thuenauer, None; Ching-Kang Chen, None; Yu-Ting Yan, None; Ching-Hwa Sung, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 574. doi:
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      Ya-Chu Hsu, Jen-Zen Chuang, Roland Thuenauer, Ching-Kang Jason Chen, Yu-Ting Yan, Ching-Hwa Sung; Endosome-dependent Rhodopsin trafficking for outer segment renewal. Invest. Ophthalmol. Vis. Sci. 2016;57(12):574.

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

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Abstract

Purpose : The outer segment (OS) of rod photoreceptors undergoes dynamic renewal on a daily basis. However, the mechanism behind this process remains to be fully understood. SARA, a Rhodopsin-binding protein, is an early endosome protein and promotes tethering and fusion of Rhodopsin vesicles during disc biogenesis. We test the hypothesis that endosomes regulated by SARA as well as GTPase Dynamin is involved in protein transport during OS renewal.

Methods : Reversible aggregation-based synchronization of Rhodopsin reporter (FM4-Rhodopsin-GFP) was transfected into Madin-Darby canine kidney (MDCK) cells and the biosynthetic pathway of Rhodopsin was traced. Dominant negative (DN) reagents of Rab11 and Dynamin were used to perturb the functions of recycling endosomes and vesicle fission events. The effects on Rhodopsin surface delivery were imaged and compared to those transfected with wildtype agents. Cre-mediated inducible expression system was employed to pulse-express wildtype or DN mutants in matured rods. Rhodopsin reporters were coexpressed to delineate the effect on Rhodopsin’s OS delivery. Rab11 expression was examined by immunolabeling in SARA-knockdown cells and Rod-specific SARA knockout retina sections. Transmission electron microscopy (EM) and focused ion beam scanning EM were employed to perform ultrastructural analyses.

Results : FM4-Rhodopsin-GFP targeted to the apical surface in polarized MDCK. Rhodopsin was traced to the Rab11+ compartments prior to arriving at the apical surface. Expression of Rab11DN or Dynamin greatly reduced the surface delivery of Rhodopsin. In vivo short-pulsed expression of Rab11DN caused Rhodopsin to mislocalize. Perturbing Dynamin functions also caused Rhodopsin to mislocalize, and we observed enlarged membrane profiles and vesicle accumulation in the inner segment. Finally, we found that rods lacking SARA had significant reduction of Rab11. SARA knockout rods had smaller OS diameters and aberrant vesicles in the OS. The structural defects were accompanied by mislocalization of Rhodopsin.

Conclusions : Our data support the hypothesis that endosomes are an integral part of the Rhodopsin transport pathway. Endosomes coupled with the fission activities of Dynamin are a source of OS-destined vesicles. Sara plays a critical role in maintaining the integrity of endosomes vital for protein trafficking and disc morphogenesis.

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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