title: The Leber congenital amaurosis protein AIPL1 and EB proteins co-localize at the photoreceptor cilium.
abstract: OBJECTIVE
  The 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).
  
  
  METHODS
  The 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.
  
  
  RESULTS
  Yeast 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.
  
  
  CONCLUSIONS
  AIPL1 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.
authors: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Juan
authors: J
authors: Hidalgo-de-Quintana
authors: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Nele
authors: N
authors: Schwarz
authors: Department of Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Ingrid P
authors: IP
authors: Meschede
authors: Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg University of Mainz, Mainz, Germany.
authors: Gabriele
authors: G
authors: Stern-Schneider
authors: Department of Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Michael B
authors: MB
authors: Powner
authors: Section of Ophthalmology and Neuroscience, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, United Kingdom.
authors: Ewan E
authors: EE
authors: Morrison
authors: Department of Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Clare E
authors: CE
authors: Futter
authors: Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg University of Mainz, Mainz, Germany.
authors: Uwe
authors: U
authors: Wolfrum
authors: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Michael E
authors: ME
authors: Cheetham
authors: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom.
authors: Jacqueline
authors: J
authors: van der Spuy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057130:Leber Congenital Amaurosis / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism*
chemical_list: C403625:AIPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D005136:Eye Proteins; D008869:Microtubule-Associated Proteins