The USH1G Protein SANS Collaborates With Cytoplasmic Dynein Motor Components In Mammalian Photoreceptor Cells

Abstract

Purpose: : Human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is associated with microtubules and mediates a transport related periciliary protein network in photoreceptor cells. The present work was designed to enlighten the function of SANS in intracellular transport in photoreceptor cells.

Methods: : Identification and validation of protein-protein interactions via yeast-2-hybrid (Y2H) screen of retinal cDNA library, GST-pull down, co-transfection assays, microtubule spin-down and microtubule destabilization assays. Investigation of protein localization in mouse and human retina by indirect immunofluorescence and immunoelectron microscopy.

Results: : In our Y2H screen we identified the interaction between SANS and dynactin-1 (p150Glued), a subunit of the dynactin complex and cargo linker to the cytoplasmic dynein motor. This interaction was validated in independent interaction assays in vitro and in cell culture. Furthermore, we demonstrated the integration of SANS into dynein transport complex by pull down of SANS with GST-fused cytoplasmic dynein intermediate chain (cDIC74). Correlative immunofluorescence and immunoelectron microscopy revealed co-localization of p150Glued, basic dynein components and SANS at microtubule tracks of photoreceptor inner segments.

Conclusions: : The present data strengthen our hypothesis that SANS participates in cargo transport to its ciliary destination. Here we demonstrate the integration of SANS into cytoplasmic dynein-dynactin complexes, essential for microtubule based transport in photoreceptor cells. Defects in these transport mechanisms may lead to retinal degeneration as characteristic for USH1G patients.

Keywords: photoreceptors • proteins encoded by disease genes • protein structure/function