The apical junction complex in cochlear basal cells

摘要 我们研究的长期目标是确定基底细胞顶端的分子组成。 交界处复合体。先前，我们证明了在基底细胞中claudin 11紧密连接是必需的。 产生耳蜗内电位并插入连接蛋白26间隙连接结构域，该连接蛋白26结构域循环K 进入内淋巴。在这个项目中，我们将识别与膜相关的细胞质蛋白， 协调装配并维护根尖连接复合体。 在初步数据中，我们显示了来自我们培育的敲门小鼠(Bira-cldn11)的活体数据， 哪种BioID技术已经通过同源重组被整合到Claudin 11基因中。 这只小鼠没有表型，听力阈值和听性脑干反应正常；因此，我们 可以得出结论，大肠杆菌Bira生物素连接酶-claudin11融合蛋白是无毒的。我们证明了核聚变 蛋白由耳蜗基底层细胞表达，并与血管纹的链霉亲和素标记共定位 在高生物素条件下，而不是在对照中。Western blotting结果表明，几种耳蜗蛋白(MR 23-35 kDa)，其中一个可能是claudin 11，在高生物素存在下被生物素化，但在对照中没有。 最后，我们给出了含有生物素的Claudin 11的C末端胰蛋白酶多肽的质谱学数据 附着在内部赖氨酸残基上的部分。该多肽是从Bira-cldn11小鼠中分离纯化出来的。 生物素条件。我们还没有在低生物素条件下从小鼠身上分离出这样的多肽。 在目标1a中，我们将从Bira-cldn11小鼠中提纯耳蜗蛋白用于质谱分析。我们将确定 克隆这些蛋白的cDNA，用于在培养的转基因细胞中进行表达分析。 在目标1b中，我们将测试确定的候选蛋白是否确实与claudin 11和claudin 11共定位 小鼠内耳组织切片中连接蛋白26的表达。我们还将产生稳定转染的MDCK细胞 表达这些蛋白和claudin 11或缝隙连接蛋白26，我们将用免疫共沉淀法将这些候选蛋白 确定它们是否拖累了claudin 11和连接蛋白26(反之亦然)。 在目标1c中，我们将使用稳定表达bira-cldn11的mdck细胞来确定我们是否可以 用质谱仪纯化和鉴定生物素化的候选蛋白。 总之，AIMS 1a-c中的实验包括对根尖连接的详细的多步分析。 基底细胞中的复杂蛋白质，可能为深入了解这一重要的 正常听力所需的复合体。

ABSTRACT 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 1a, 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 1b, 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 1c, 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.