IDENTIFICATION OF MAMMALIAN INTRAFLAGELLAR TRANSPORT COMPLEXES FOR PRIMARY CILIA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 初级纤毛在多种发育和病理过程中起着重要但尚未明确的作用。 这些功能的核心是激活细胞并产生基于微管的投射的能力。 在绿色藻类莱茵衣藻中，鞭毛内运输是鞭毛建立和维持的过程。 一些哺乳动物等效物现已显示在体内执行类似的功能，并产生病理学，例如在多种人类疾病中观察到的多指畸形、囊性肾和退行性失明（例如Bardet-Beidl综合征、多囊肾病和肾病）。 到目前为止，还没有一个明确的蛋白质组学目录的鞭毛内运输机制在哺乳动物细胞。 此外，这种运输机制也将与运往纤毛的货物（如受体、信号蛋白和结构因子）以及负责运输过程的马达联系起来。 为此，我们已经产生了鼠肾上皮细胞系，其以高倾向产生纤毛，稳定表达与IFT组分的串联亲和融合。 该细胞系将用于产生大量材料，从中纯化IFT复合物并通过质谱法进行分析。 在蛋白质组学分析的同时，我们还进行了显微镜分析，以可视化鞭毛内运输的过程，并确定运输的动力学及其与建立初级纤毛及其维持的关系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Primary cilia play an important, but as yet, undefined role in a wide variety of developmental and pathological processes. Central to these functions is the ability to polarize a cell and produce the microtubule-based projection. In the green algae, Chlamydomonas Reinhardtii, a process termed intraflagellar transport is responsible for the establishing and maintaining flagella. Some of the mammalian equivalents have now been shown to perform a similar function in vivo and produce pathologies such as polydactyly, cystic kidneys and degenerative blindness seen in a variety of human diseases (e.g. Bardet-Beidl syndrome, Polycystic Kidney disease and Nephronophthsis). As yet, there does not exist a well-defined proteomic catalogue of the intraflagellar transport machinery in mammalian cells. Moreover, this transport machinery will also be linked to cargos destined for the cilium, such as receptors, signaling proteins and structural factors, and the motors responsible for the transport process. To this end, we have generated a murine kidney epithelial cell line, one that produces cilia at high propensity, stably expressing a tandem affinity fusion to an IFT component. This cell line will be used to generate substantial material from which IFT complexes will be purified and analyzed by mass spectrometry. In parallel with the proteomic analysis, we are also carrying out microscopic analyses to visualize the process of Intraflagellar Transport and identify the dynamics of transport and their relation to establishing the primary cilium and its maintenance.