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Uwe Wolfrum, Nasrin Sorusch, Karsten Boldt, Marius Ueffing, Kirsten A. Wunderlich; The USH1G scaffold protein SANS is part of the cytoplasmic dynein transport module in the photoreceptor inner segments. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3171.
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© ARVO (1962-2015); The Authors (2016-present)
Human Usher syndrome (USH) is the most common form of combined deaf-blindness and a complex disease. To get insights into the molecular function of the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) we aim to identify protein interactions that disclose functional modules related to USH protein complexes in retinal photoreceptor cells.
We identified proteins and complexes interacting with SANS by yeast-2-hybrid screens (Y2H) of retinal cDNA library, affinity proteomics based on tandem-affinity purification, and co-immunoprecipitation from HEK293T cells and retinal tissue, respectively. We validated interactions by complementary pull down- and co-transfection assays. We showed subcelluar localization of complex partners in retinal photoreceptor cells by light and electron microscopy and applied proximity ligation assays (PLA) to demonstrate complexes in situ. We applied shRNAs and siRNAs for protein depletion from cells. We analysed transport modules by a fluorescence recovery after photo bleaching (FRAP)-based method in living photoreceptor cells.
We demonstrated SANS in protein complexes together with several components of the cytoplasmic dynein motor module, regulatory small GTPases, and rhodopsin. sh/siRNA knock-downs of the complex components led to the decrease of the number of primary cilia in starved cells. Immunocytochemistry and PLAs revealed the localization of SANS related protein complexes along microtubule tracks in the inner segment and the periciliary region at the base of photoreceptor cilia. FRAP experiments demonstrated that the apical transport of rhodopsin to the photoreceptor cilium is microtubule dependent in vivo.
Our data support that the USH1G protein SANS participates at the microtubule-dependent vesicular transport of ciliary molecules, e.g. rhodopsin mediated by cytoplasmic dynein. Defects in this transport module lead to ciliary dysfunction and photoreceptor cell death which may underlay the retinal degeneration as characteristic for USH1G patients.
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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