The Molecular Arrangement of on Usher Syndrome Protein Network at the Photoreceptor Cilium and Its Role in the Intersegmental Transport in Photoreceptors

Abstract

Purpose:: The Human Usher syndrome (USH) is the most common form of combined deaf-blindness. USH is clinical (USH type 1 to 3) and genetically heterogeneous and 10 USH genes encode proteins of diverse protein families. All identified USH type 1/2 proteins can be connected by the scaffold protein harmonin (USH1C) within a USH interactom. Here, we address the question whether USH1/2 proteins are also qualified to form a protein network at the ciliary apparatus of retinal photoreceptors in the absence of harmonin.

Methods:: Subcellular localization of proteins: Western blots of retinal tangential sections and subcellular fractionation, immunofluorescence and immunoelectron microscopy of mouse and Xenopus retinas; protein-protein interaction assays: yeast-two-hybrid, co-transfection of cell lines, GST-pull down assays of recombinant expressed polypeptides; pharmacological microtubule destabilization in organotypic retina cultures.

Results:: In retinal photoreceptors, the subcellular distribution of SANS (USH1G) was dependent on the microtubule cytoskeleton. SANS co-localized with USH2A, VLGR1 (USH2C), and the scaffold protein whirlin in the ciliary apparatus - especially in the periciliary ridge complex of photoreceptor cells. In this complex, USH2 proteins provide molecular linkages between the plasma membrane of the cilium and the inner segment (calycal processes) of photoreceptor cells. Whirlin directly bound SANS and the USH2 proteins via its PDZ-domains.

Conclusions:: In the ciliary specialization of photoreceptor cells, USH proteins are integrated into a protein network, associated with the cytoskeleton. As molecular components of the periciliary ridge complex this network may contribute to the handover of cargos between the inner segment and the transport through the connecting cilium. Defects of complex partners may lead to dysfunction of the entire USH network and cause photoreceptor degeneration in USH patients.

Keywords: photoreceptors • retinal degenerations: cell biology • retinal degenerations: hereditary