Mechanism of mTOR Signaling to Autophagy Machinery

描述（由申请人提供）：本修订申请的目的是通过使用尖端细胞成像技术结合TALEN技术分析自噬体形成早期步骤中发生的蛋白质-蛋白质相互作用来扩展母基金的范围和能力。自噬体早期膜结构（隔离膜）形成的分子机制至今仍是一个谜，是细胞生物学中的一个基本问题。隔离膜开始从内质网中出现，并扩展形成包围捕获的大分子的双膜囊泡结构，称为自噬体。我的小组最近的研究表明，mTORC 1，ULK 1-Atg 13-FIP 200-Atg 101复合物和Beclin 1-PI 3 KC 3（hVps 34）-Atg 14 L复合物相互作用，并以依赖于营养素和mTORC 1活性的方式形成大分子相互作用。目前尚不清楚多蛋白复合物之间的相互作用，以调节隔离膜的形成。这个领域中备受关注的问题必须通过革命性的细胞成像工具来解决。细胞成像技术不断发展，在研究中使用尖端工具非常重要。在母基金中，我们提出了三个具体目标：（1）确定mTORC 1如何负调控ULK 1-Atg 13-FIP 200-Atg 101复合物;（2）确定Atg 13在自噬诱导中的作用;（3）确定ULK 1如何调节含Atg 14 L的PI 3 KC 3复合物。在这个修改后的应用程序中，我们增加了新的细胞成像工具和遗传工具，以加强每个目标。我们将使用显微镜工具进行高分辨率活细胞成像，FLIM/FRET和nanoscope技术来研究自噬蛋白复合物如何以分层方式招募，以及它们如何在自噬体形成过程中邻近共定位，以及相互作用如何通过自噬蛋白的磷酸化来调节。本修订研究包括TALEN技术，以生成KO和KI细胞系。这种强大的遗传工具对于产生细胞系统以研究翻译后修饰（如磷酸化）、克服使用重组蛋白过表达的问题将是必不可少的。正如其在许多细胞系统中的强大，有效的应用所证明的那样，TALEN技术将在自噬领域非常有用，可以解剖自噬途径。在这个修订后的应用程序中引入的新工具将解决以前看不见的蛋白质-蛋白质相互作用，特别是动态相互作用，并以分层方式定义自噬蛋白的招募，从而使我们能够更好地了解自噬体形成的过程和机制。

DESCRIPTION (provided by applicant): The objective of this revision application is to extend the scope and capabilities of the parent grant by analyzing the protein-protein interactions occurring in the early steps of autophagosome formation using cutting-edge cell imaging techniques combined with TALEN technology. The molecular mechanisms underlying the formation of the early membrane structure of autophagosome called the isolation membrane remains mystery and it is a fundamental question in cell biology. The isolation membrane starts to emerge from the endoplasmic reticulum, and is expanded to form the double-membrane vesicular structure called autophagosome that encircles captured macromolecules. Recent studies from my group revealed that mTORC1, the ULK1-Atg13-FIP200-Atg101 complex, and the Beclin 1-PI3KC3 (hVps34)-Atg14L complex interact and form a large macromolecular interaction in a manner dependent upon nutrients and mTORC1 activity. It remains unclear what interactions are involved between the multi-protein complexes to regulate the isolation membrane formation. The well-appreciated question in the field must be greatly addressed by revolutionary cell imaging tools. Cellular imaging techniques continuously evolve and it is important to implement cutting-edge tools in the study. In the parent grant, we proposed three specific aims: (1) to determine how mTORC1 negatively regulates the ULK1-Atg13-FIP200-Atg101 complex; (2) to define the role of Atg13 in autophagy induction; (3) to determine how ULK1 regulates the Atg14L-containing PI3KC3 complex. In this revised application, we have added new cell imaging tools and genetic tools to strengthen each aim. We will use microscopic tools for high-resolution live cell imaging, FLIM/FRET and nanoscope techniques to investigate how the autophagy protein complexes are recruited in a hierarchical manner and how they colocalize in proximity during the autophagosome formation and how the interactions are regulated by phosphorylation of autophagy proteins. This revised study includes the TALEN technology to generate KO and KI cell lines. This powerful genetic tool will be essential to generate cellular systems to study post- translational modifications, such as phosphorylation, overcoming the concern with use of recombinant protein overexpression. As demonstrated in its powerful, efficient applications in many cellular systems, the TALEN technology will be very useful in autophagy field in dissecting the autophagy pathway. The new tools introduced in this revised application will resolve previously-unseen protein-protein interactions, especially dynamic interactions, and define the recruitment of autophagy proteins in a hierarchical manner, thus enabling us to better understand the processes and mechanisms of the formation of autophagosome.