Mechanisms of membrane trafficking in endocytic and non-endocytic pathways

项目摘要/摘要 受体的内化和再循环是所有真核细胞的关键生物学过程。这个 早期/分选内含体是从质膜(PM)内化的受体的初始目的地。这 内体是一个主要的分拣站，受体从这里分流到晚期的内体和 溶酶体用于降解，或通过囊泡和短暂性网络循环回到PM 管状再循环型内小体。而十年前，大多数研究人员认为主动排序直接 蛋白质到降解途径，靶向到循环途径，被认为在很大程度上是被动的 默认情况下发生的进程。然而，最近的证据支持通过以下方式对回收途径进行主动分类 特异性分选Nexin(SNX)和其他与受体细胞质尾部结合的蛋白质和特异性 把它们作为回收的目标。尽管回收是所有哺乳动物细胞的基本过程，但它的复杂性 调控机制知之甚少，包括内体膜上受体的分类、萌发和 内小体的囊泡和小管的分裂，以及受体回到PM的运输因此，我们的 对内体功能的认识大大落后于受体内化机制。一把钥匙 控制内体分选和运输的一组调节蛋白质是逆转录复合体。 最初在从内体到高尔基复合体的生物合成货物的检索中被鉴定，逆转录聚体 最近参与了多种关键细胞通路的调节，包括细胞内和细胞外 胞内转运的范围包括胞内循环、线粒体动态平衡、中心体循环 纤毛发生。该反转录复合体还与其他关键的内吞调节蛋白相互作用，包括 管状内体支架Mical-L1；其相互作用伙伴和内体分裂调节剂，EH结构域 包含1(EHD1)；和许多介导内体货物分选的SNX蛋白。逆转录病毒还 通过WASH复合体连接到肌动蛋白细胞骨架。我们的实验室一直致力于研究一种 了解内吞调节蛋白在两种内吞途径中的作用机制 以及在非内吞细胞交易中。我们的主要专长是生物化学和分子细胞生物学耦合。 使用先进的光学显微镜，但我们认识到有必要将体内成分结合到我们的 方法并与其他小组进行持续合作，以在整个生物体中检查这些过程， 包括斑马鱼和蠕虫。在我们的研究中，我们将解决重要的和尚未解决的生物学问题 问题如：1)如何调节内体分裂并将其与分类和回收联系起来；2)如何关键 内吞蛋白参与原生纤毛的生物发生。

PROJECT SUMMARY/ABSTRACT The internalization and recycling of receptors is a key biological process in all eukaryotic cells. The early/sorting endosome is the initial destination of receptors internalized from the plasma membrane (PM). This endosome serves as a major sorting station from which receptors are shunted to late endosomes and lysosomes for degradation, or are recycled back to the PM through a transitory network of vesicular and tubular recycling endosomes. Whereas a decade ago most researchers thought that active sorting directed proteins to the degradation pathways, targeting to the recycling pathway was thought to be largely a passive process that occurs by default. However, recent evidence supports active sorting to the recycling pathways by specific sorting nexin (SNX) and other proteins that bind to the cytoplasmic tails of receptors and specifically target them for recycling. Although recycling is an essential process for all mammalian cells, its complex regulation is poorly understood including the sorting of receptors on endosomal membranes, the budding and fission of vesicles and tubules from the endosome, and the transport of receptors back to the PM. As such, our knowledge of endosomal function lags substantially behind that of receptor internalization mechanisms. A key group of regulatory proteins that controls sorting and trafficking at the endosome is the retromer complex. Originally identified in the retrieval of biosynthetic cargo from endosomes to the Golgi complex, the retromer has recently been implicated in the regulation of a variety of key cellular pathways both within and beyond the scope of endocytic trafficking including endocytic recycling, mitochondrial homeostasis, the centrosome cycle and ciliogenesis. The retromer complex also interacts with other key endocytic regulatory proteins, including the tubular endosome scaffold MICAL-L1; its interaction partner and endosomal fission modulator, EH domain containing 1 (EHD1); and a host of SNX proteins that mediate endosomal cargo sorting. The retromer also links to the actin cytoskeleton via the WASH complex. Our laboratory has been focusing on an overall understanding of the mechanisms by which endocytic regulatory proteins function both in endocytic pathways and in non-endocytic trafficking. Our primary expertise is in biochemistry and molecular cell biology coupled with advanced light microscopy, but we recognize the need to incorporate in vivo components into our approach and have ongoing collaborations with other groups to examine these processes in whole organisms, including zebrafish and worms. In our studies, we will address significant and as-yet-unresolved biological problems such as: 1) how endosomal fission is regulated and linked to sorting and recycling and 2) how key endocytic proteins mediate the biogenesis of the primary cilium.