The Leber congenital amaurosis protein AIPL1 and EB proteins co-localize at the photoreceptor cilium.

Juan Hidalgo-De-Quintana,Gabriele Stern-Schneider,Michael E Cheetham,Uwe Wolfrum,Ewan E Morrison,Nele Schwarz,Ingrid P Meschede,Jacqueline Van Der Spuy,Michael B Powner,Clare E Futter,Tiansen Li

doi:10.1371/journal.pone.0121440

Juan Hidalgo-De-Quintana, Gabriele Stern-Schneider + Show 9 more

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https://doi.org/10.1371/journal.pone.0121440

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Abstract

PurposeThe aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).MethodsThe CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.ResultsYeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.ConclusionsAIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.

Highlights

Mutations in the aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) gene cause the devastating disease Leber’s congenital amaurosis (LCA) [1], which is characterized by profound visual impairment or loss at birth
Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, end-binding protein 1 (EB1) and EB3
Immunofluorescent confocal microscopy revealed that AIPL1 did not colocalize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin

Summary

Introduction

Mutations in the AIPL1 gene cause the devastating disease Leber’s congenital amaurosis (LCA) [1], which is characterized by profound visual impairment or loss at birth. FKBP52 potentiates transcriptional activity via a dual mechanism involving the increased ligand-binding affinity of the Hsp90-associated receptor, and by targeting the efficient microtubule-dependent retrotranslocation of the signalling complex mediated by the direct interaction of FKBP52 with the dynein molecular motor [4,5,6,7,8]. This mechanism is facilitated by the direct interaction of FKBP52 with tubulin thereby linking the heterocomplex to cytoskeletal tracts, and an FKBP52 microtubule depolymerization activity has revealed a role in microtubule dynamics [9,10,11]. AIP, similar to FKBP51 and FKBP52, modulates the transcriptional activity of the Hsp90-bound aryl hydrocarbon receptor [14]

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