Cytoskeletal Regulation Of Vesicle Transport In Liver

描述(由申请人提供)：本提案的总体目标是定义支持肝细胞中受体介导的囊泡运输过程的分子机制。这些过程对于正常的铁稳态、生长因子受体信号转导和细胞存活是必不可少的。此外，它们的破坏可能导致各种流行的肝病。在这里，我们主要关注肝细胞在质膜(PM)和跨高尔基网络(TGN)上利用基于囊泡的机制来分类和运输不同的受体和货物蛋白。这些包括转铁蛋白受体(TFR)、表皮生长因子受体(EGFR)和用于降解它们的溶酶体水解酶。我们有大量的初步数据表明，肝细胞中一种保守的受体分选和囊泡形成机制的作用，该机制以膜切断GTP酶Dynamin 2(Dy2)为中心。我们发现，这种可收缩的聚合物与肌动蛋白细胞骨架和特定的接头蛋白结合在一起，调节受体的内吞作用，并与TGN结合，介导新生水解酶向溶酶体的运输。最近，我们发现这个复合体可以通过与泛素相关蛋白的相互作用来分拣货物，并受非受体酪氨酸激酶的调节。因此，这一提议的中心假设是，多聚体Dy2收缩支架与肌动蛋白细胞骨架和特定的接头蛋白结合，介导肝细胞中PM和TGN的货物隔离和囊泡形成的双重过程。我们将使用最先进的成像、生化和分子方法来探索三个相关但独立的目标。在第一个目标中，我们将测试Dy2-肌动蛋白内吞复合体的调节组装如何介导来自肝细胞质膜的两种不同TFR的不同内化和转运在第二个目标中，我们将定义Dy2复合体如何与肝细胞中基于泛素的分选机制相互作用，以介导EGFR的内化和内吞后转运，将该受体靶向溶酶体进行降解。最后，在第三个目标中，我们将定义Dy2复合体如何与肝细胞TGN上基于泛素的新型分选机制相互作用，以维持高尔基体结构，并介导新合成的水解酶运输到溶酶体以降解EGFR。这一提议在概念和技术上都是创新的，因为它为理解必需的肝细胞受体和货物蛋白如何被差异地分选到囊泡载体以便随后运输到不同的细胞器的分子机制提供了重大进展。这些研究收集的信息将有助于确定治疗和预防影响基本肝功能的疾病的策略。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the molecular machinery that supports receptor-mediated vesicle trafficking processes in hepatocytes. These processes are essential for normal iron homeostasis, growth factor receptor signaling, and cell survival. Further, their disruption can lead to a variety of prevalent liver diseases. Here we focus on the vesicle-based machinery utilized by the hepatocyte at the plasma membrane (PM) and trans-Golgi network (TGN) to sort and traffic distinct receptors and cargo proteins. These include the transferrin receptors (TfRs), the epidermal growth factor receptor (EGFR), and lysosomal hydrolases used in their degradation. We have substantial preliminary data implicating the action of a conserved receptor sorting and vesicle formation machinery in hepatocytes that is centered around the membrane-severing GTPase dynamin 2 (Dyn2). We have found that this contractile polymer associates with the actin cytoskeleton and specific adaptor proteins at both the PM to regulate receptor endocytosis and the TGN to mediate trafficking of nascent hydrolases to the lysosome. Most recently, we have found that this complex can sort cargo through interactions with ubiquitin-associated proteins and is regulated by non- receptor tyrosine kinases. Thus, the CENTRAL HYPOTHESIS of this proposal is that a polymeric Dyn2 contractile scaffold associates with the actin cytoskeleton and specific adaptor proteins to mediate the dual processes of cargo sequestration and vesicle formation from the PM and TGN in hepatocytes. We will use state of the art imaging, biochemical, and molecular methods to explore three related but independent aims. In Aim One we will test how the regulated assembly of a Dyn2-actin endocytic complex mediates differential internalization and trafficking of the two different TfRs from the hepatocyte PM. In Aim Two we will define how the Dyn2 complex interacts with the ubiquitin-based sorting machinery in hepatocytes to mediate internalization and post-endocytic trafficking of the EGFR, targeting this receptor to lysosomes for degradation. Finally, in Aim Three we will define how the Dyn2 complex interacts with a novel ubiquitin-based sorting machinery at the hepatocyte TGN to maintain Golgi structure and mediate transport of newly synthesized hydrolases to the lysosome for EGFR degradation. This proposal is conceptually and technically innovative in that it provides major advances toward understanding the molecular mechanism of how essential hepatocyte receptors and cargo proteins are differentially sorted into vesicle carriers for subsequent trafficking to distinct organelles. The information gathered by these studies will help identify strategies for the treatment and prevention of diseases that affect basic liver function.