The apical junction complex in cochlear basal cells

摘要 我们研究的长期目标是表征基底细胞顶端的分子组成， 连接复合体以前，我们证明了基底细胞中的claudin 11紧密连接是必要的， 产生耳蜗内电位和插入连接蛋白26间隙连接结构域，其再循环K+ 进入内淋巴在这个项目中，我们将确定膜相关的细胞质蛋白， 协调组装并维持顶端连接复合体。 在初步数据中，我们显示了来自我们产生的敲入小鼠（BirA-cldn 11）的体内数据， 该BioID技术已经使用同源重组被并入Claudin 11基因中。 这只小鼠没有表型，听力阈值和听觉脑干反应正常;因此，我们 可以得出结论，大肠杆菌BirA生物素连接酶-紧密连接蛋白11融合蛋白没有毒性。我们发现， 蛋白质由耳蜗基底细胞表达，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。我们通过蛋白质印迹法表明，几种耳蜗蛋白（Mr 23- 35 kDa），其中之一可能是紧密连接蛋白11，在高生物素存在下被生物素化，但在对照中没有。 最后，我们显示了含有生物素的claudin 11的C-末端胰蛋白酶肽的质谱数据。 连接到内部赖氨酸残基的部分。该肽在高浓度下从BirA-cldn 11小鼠中纯化。 生物素条件我们还没有从低生物素条件下的小鼠中分离出这样的肽。 在目标1a中，我们将从BirA-cldn 11小鼠中纯化耳蜗蛋白用于质谱分析。我们将确定 克隆这些蛋白质的cDNA，用于培养中转染细胞的表达分析。 在目标1b中，我们将测试所鉴定的候选蛋白质是否实际上与claudin 11共定位， 小鼠内耳组织切片中的连接蛋白26。我们还将产生稳定转染的MDCK细胞， 表达这些蛋白质和紧密连接蛋白11或连接蛋白26，我们将这些候选物共免疫沉淀， 确定它们是否拉低了紧密连接蛋白11和连接蛋白26（反之亦然）。 在目标1c中，我们将使用表达BirA-cldn 11的稳定转染的MDCK细胞来确定我们是否可以 通过质谱法纯化和鉴定生物素化的候选蛋白。 总之，目的1a-c中的实验包括对顶连接区的详细多步分析。 复杂的蛋白质在基底细胞，这将可能提供深入了解组装和功能，这一重要的 这是正常听力所必需的。

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.