SANS (USH1G) expression in developing and mature mammalian retina.

The 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 prepubertal-onset of retinitis pigmentosa. Five corresponding genes of the six USH1 genes have been cloned so far. The USH1G gene encodes the SANS (scaffold protein containing ankyrin repeats and SAM domain) protein which consists of protein motifs known to mediate protein-protein interactions. Recent studies indicated SANS function as a scaffold protein in the protein interactome related to USH. Here, we generated specific antibodies for SANS protein expression analyses. Our study revealed SANS protein expression in NIH3T3 fibroblasts, murine tissues containing ciliated cells and in mature and developing mammalian retinas. In mature retinas, SANS was localized in inner and outer plexiform retinal layers, and in the photoreceptor cell layer. Subcellular fractionations, tangential cryosections and immunocytochemistry revealed SANS in synaptic terminals, cell-cell adhesions of the outer limiting membrane and ciliary apparati of photoreceptor cells. Analyses of postnatal developmental stages of murine retinas demonstrated SANS localization in differentiating ciliary apparati and in fully developed cilia, synapses, and cell-cell adhesions of photoreceptor cells. Present data provide evidence that SANS functions as a scaffold protein in USH protein networks during ciliogenesis, at the mature ciliary apparatus, the ribbon synapse and the cell-cell adhesion of mammalian photoreceptor cells. Defects of SANS may cause dysfunction of the entire network leading to retinal degeneration, the ocular symptom characteristic for USH patients.

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.