June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
ClC-2 chloride channel localizes to the apical surface and primary cilium of RPE cells and regulates ciliogenesis
Author Affiliations & Notes
  • Enrique J. Rodriguez-Boulan
    Ophthalmology-Dyson Vision Research Inst, Weill Cornell Medical College, New York, New York, United States
  • Ignacio Benedicto
    Ophthalmology-Dyson Vision Research Inst, Weill Cornell Medical College, New York, New York, United States
  • Erwin de la Fuente
    Facultad de Medicina, Universidad Catolica del Norte, Coquimbo, Chile
  • Diego Gravotta
    Ophthalmology-Dyson Vision Research Inst, Weill Cornell Medical College, New York, New York, United States
  • Agustin Anastasia
    INIMEC-CONICET, Cordoba, Argentina
  • Zelda Salfati
    Ophthalmology-Dyson Vision Research Inst, Weill Cornell Medical College, New York, New York, United States
  • Guillermo Lehmann-Mantaras
    Ophthalmology-Dyson Vision Research Inst, Weill Cornell Medical College, New York, New York, United States
  • Footnotes
    Commercial Relationships   Enrique Rodriguez-Boulan, None; Ignacio Benedicto, None; Erwin de la Fuente, None; Diego Gravotta, None; Agustin Anastasia, None; Zelda Salfati, None; Guillermo Lehmann-Mantaras, None
  • Footnotes
    Support  NIH/NEI R01 EY08538
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 358. doi:
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    • Get Citation

      Enrique J. Rodriguez-Boulan, Ignacio Benedicto, Erwin de la Fuente, Diego Gravotta, Agustin Anastasia, Zelda Salfati, Guillermo Lehmann-Mantaras; ClC-2 chloride channel localizes to the apical surface and primary cilium of RPE cells and regulates ciliogenesis
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):358.

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

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Abstract

Purpose : Chloride channel 2 (ClC-2) is a ubiquitous polytopic membrane protein localized to the basolateral plasma membrane of many body epithelia that plays important physiological roles in cell volume regulation, ion transport and acid-base regulation. ClC-2 knock-out in mice causes retinal degeneration and blindness. Here, we report an unexpected apical and Primary Cilium (PC) localization of ClC-2 in RPE cells.

Methods : We performed confocal immune-fluorescence microscopy to localize ClC-2-HA in polarized ARPE-19 and hfRPE cells transduced with lentiviral vectors. To explore the localization of endogenous ClC-2 we carried out domain-specific cell surface biotinylation assays. To examine the localization of the channel in mouse RPE in vivo, we performed subretinal injection of a plasmid encoding ClC-2-GFP in wild type C57BL/6 mice. Ciliogenesis was induced in cultured ARPE-19 cells by serum starvation for 48h in control and lentivirus-transduced cells with shRNA against CLC-2. To activate the Hedgehog (Hh) pathway, we exposed ARPE-19 cells to 100nM Smoothened agoinst (SAG), a Hh agonist, for 48h.

Results : Surprisingly, in striking contrast with previous physiological studies, our immunofluorescence and surface biotinylation experiments demonstrate that ClC-2 is predominantly localized to the apical surface and PC of cultured RPE cells from different sources. This result was confirmed in vivo by subretinal electroporation of ClC-2-GFP into mice eyes. The apical and PC localization appears to be cell-specific, as there is no detectable signal for ClC-2-HA in the PC of MDCK cells, where ClC-2 localizes basolaterally. Quantification of the percentage of ciliated cells and cilium length showed that ClC-2 depletion significantly impairs cilia formation. Because PC is home for the Hh pathway, we exposed ARPE-19 cells to a Hh agonist (SAG) and found that ClC-2 knockdown attenuates Hh signaling, consistent with a decreased population of ciliated RPE cells.

Conclusions : We show for the first time that ClC-2 is predominantly enriched in the PC of RPE cells, where it seems to play a role in the genesis of primary cilia. We are now exploring additional physiological roles of apical ClC-2 in RPE cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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