The long term goal of our research is to characterize the molecular composition of the basal cell apical junction complex. Previously, we demonstrated that claudin 11 tight junctions in basal cells are necessary for generating the endocochlear potential and for intercalating connexin 26 gap junction domains, which recycle K+ into the endolymph. In this project, we will identify membrane-associated cytoplasmic proteins that recruit, coordinate the assembly and maintain the apical junctional complex. In the preliminary data, we show in vivo data from a knockin mouse we generated (BirA-cldn11), in which BioID technology has been incorporated into the Claudin 11 gene using homologous recombination. This mouse has no phenotype, and hearing thresholds and auditory brainstem responses are normal; thus, we can conclude that the E.coli BirA biotin ligase-claudin 11 fusion protein is not toxic. We show that the fusion protein is expressed by cochlea basal cells and colocalizes with streptavidin labeling of the stria vascularis under high biotin conditions but not in controls. We show by western blotting that several cochlear proteins (Mr 23-35kDa), one of which is likely claudin 11, are biotinylated in the presence of high biotin but not in controls. Finally, we show mass spectrometry data for a C-terminal tryptic peptide of claudin 11, containing a biotin moiety attached to an internal lysine residue. This peptide was purified from a BirA-cldn11 mouse under high biotin conditions. We have not isolated such a peptide from mice under low biotin conditions.
In Aim 1 a, we will purify cochlear proteins from BirA-cldn11 mice for mass spectrometry. We will identify an clone the cDNAs for these proteins for expression analysis in transfected cells in culture.
In Aim 1 b, we will test whether the candidate proteins identified actually colocalize with claudin 11 and connexin 26 in tissue sections from mouse inner ear. We will also generate stably transfected MDCK cells expressing these proteins and claudin 11 or connexin 26 and we will co-immunoprecipitate these candidates to determine if they pulldown claudin 11 and connexin 26 (and vice versa).
In Aim 1 c, we will use stably transfected MDCK cells expressing BirA-cldn11 to determine if we can purify and identify biotinylated candidate proteins by mass spectroscopy. Together, the experiments in Aims 1a-c comprise a detailed multi-step analysis of apical junctional complex proteins in basal cells that will likely provide insight into the assembly and function of this important complex that is required for normal hearing.
In this proposal, we will characterize components of a macromolecular complex in the cochlea known as the basal cell apical junction complex. Our work over the past 20 years demonstrates how and why this complex is critical for normal hearing in mice with direct application to familial forms of deafness in humans.
