Intercellular Signaling and Endosome to Golgi Transport in Multicellular Animals

描述(由申请人提供)：我们在这项提案中的长期目标是识别和了解将内吞体内的货物从内体运输到高尔基体的细胞成分(逆行循环)，并了解这一过程如何调节，以及如何整合到细胞间信号通路中。最近，逆行循环与多种后生动物特有的过程有关，如Wnt信号梯度的产生，神经系统中AMPA受体信号的产生，死亡细胞的清除，以及上皮极性的调节。WNT信号需要内体到高尔基体的WNT配体伴侣MIG-14/Wnless的回收，而MIG-14/Wnless循环的分子细节在线虫、果蝇和哺乳动物细胞中被证明是高度保守的。如果没有逆行循环，跨膜货物蛋白，如MIG-14/Wnless，会异常进入溶酶体并被降解，从而耗尽细胞中此类蛋白的功能池。为了对逆行循环如何发挥作用以及它如何影响多细胞动物的细胞间信号机制有新的见解，我们率先对线虫的极化肠道上皮进行了分析。在新的前期工作中，我们发现在逆行循环突变体中，转化生长因子β-信号转导受损。我们计划进一步测试转化生长因子受体是逆行货物的假设，并在果蝇和哺乳动物细胞中测试这一机制的系统发育保守性。此外，我们的团队还发现了对内体调节蛋白的先前未被怀疑的要求，超出了研究得很好的逆转录复合体。这些新的调节子包括J-结构域的辅助伴侣RME-8。我们计划测试这一假设，即RME-8调控内体扁平网壳蛋白晶格的分解，在逆转录和ESCRT驱动的降解机制之间调解反馈环，该机制存在于相同的内体上。此外，我们还鉴定了参与MIG-14/Wnless逆行循环的整个额外蛋白质复合体。这种蛋白质复合体似乎与循环内小体有关，并包含膜结合、膜弯曲和肌动蛋白成核的模块。我们计划确定这种蛋白质复合体如何影响MIG-14/Wnless循环，测试它影响包括转化生长因子受体在内的其他逆行货物的能力，并测试哺乳动物细胞的功能保守性。我们预计，我们的发现将为运输机制如何影响信号通路提供独特的见解，并将提高我们治疗因受体信号强度调节不当而引起的各种疾病的能力，例如癌症，以及其他与逆行循环调节不当相关的疾病，包括阿尔茨海默氏症和帕金森氏症。

DESCRIPTION (provided by applicant): Our long-term goal in this proposal is to identify and understand the cellular components that transport internalized endocytic cargo from endosomes to the Golgi apparatus (retrograde recycling), and to understand how this process regulates, and is integrated within, intercellular signaling pathways. Retrograde recycling has recently been linked to multiple metazoan-specific processes such as the generation of Wnt signaling gradients, AMPA-receptor signaling in the nervous system, clearance of dead apoptotic cells, and regulation of epithelial polarity. Wnt signaling requires endosome-to-Golgi retrieval of the Wnt ligand chaperone MIG-14/Wntless, and the molecular details of MIG-14/Wntless recycling have proven to be highly conserved in C. elegans, Drosophila, and mammalian cells. Without retrograde recycling, transmembrane cargo proteins such as MIG-14/Wntless aberrantly enter the lysosome and are degraded, thus depleting the functional pool of such proteins from the cell. To gain novel insight into how retrograde recycling functions, and how it influences intercellular signaling mechanisms in multicellular animals, we have pioneered analysis of the polarized intestinal epithelium of C. elegans. In novel preliminary work we discovered that TGF?-signaling is impaired in retrograde recycling mutants. We plan to further test the hypothesis that TGF?-receptors are retrograde cargo, and test phylogenetic conservation of this mechanism in Drosophila and mammalian cells. In addition, our team has discovered previously unsuspected requirements for endosome regulatory proteins beyond the well-studied retromer complex. These novel regulators include the J-domain co-chaperone RME-8. We plan to test the hypothesis that RME-8 regulates the disassembly of the endosomal flat clathrin lattice, mediating a feedback loop between retromer and the ESCRT-driven degradation machinery that is present on the same endosomes. In addition we have identified an entire additional protein complex involved in retrograde recycling of MIG-14/Wntless. This protein complex appears to be associated with the recycling endosome, and contains modules for membrane binding, membrane bending, and actin nucleation. We plan to determine how this protein complex influences MIG-14/Wntless recycling, test its ability to influence other retrograde cargo including TGF?-receptors, and test for conservation of function in mammalian cells. We expect that our findings will provide unique insight into how trafficking mechanisms influence signaling pathways, and will improve our ability to treat diverse diseases that arise from misregulation of receptor signaling strength, such as in cancers, and other diseases associated with misregulated retrograde recycling including Alzheimer's and Parkinson's diseases.