IDENTIFICATION OF MAMMALIAN INTRAFLAGELLAR TRANSPORT COMPLEXES FOR PRIMARY CILI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。初级纤毛在多种发育和病理过程中发挥着重要但尚未明确的作用。这些功能的核心是极化细胞并产生基于微管的投影的能力。在绿藻中，衣藻是一种被称为鞭毛内运输的过程，负责建立和维持鞭毛。一些哺乳动物的等价物现在已被证明在体内执行类似的功能，并产生各种人类疾病(如Bardet-Beidl综合征、多囊肾疾病和肾病)中出现的多指、囊性肾脏和退行性失明等病理现象。到目前为止，还没有一个明确的哺乳动物细胞鞭毛内运输机制的蛋白质组学目录。此外，这种运输机制还将与运往纤毛的货物联系在一起，如受体、信号蛋白和结构因子，以及负责运输过程的马达。为此，我们建立了一种小鼠肾上皮细胞系，这种细胞系能够产生高倾向的纤毛，稳定地表达与IFT成分的串联亲和力融合。该细胞系将被用来产生大量的材料，从中提纯IFT复合体并用质谱仪进行分析。在蛋白质组学分析的同时，我们还进行了显微分析，以可视化鞭毛内运输的过程，并确定运输的动力学及其与初级纤毛的建立和维持的关系。

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.