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Toward Atomic Resolution of Membranes and Membrane-Associated Machines

膜和膜相关机器的原子分辨率

基本信息

批准号：
9117230
负责人：
Adam Frost
金额：
$ 170.65万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9117230
关键词：
Toward Atomic Resolution Membranes Membrane

项目摘要

DESCRIPTION (provided by applicant): The unifying goal of this proposal is to understand the structures and functions of machines that assemble on cellular membranes. Cells depend upon these machines in order to transform the shape, size and connectivity of their membranes and such "remodeling" underlies cell division, migration, differentiation, and communication. In addition, every pathogen hijacks or disrupts membrane-associated complexes to infect or escape from cells. Despite such central importance we lack comprehensive models of how membrane-binding proteins transduce signals, oligomerize, or remodel the size, shape and topology of cellular membranes. To overcome the intrinsic challenges in studying multi-component complexes that assemble transiently on membranes, we will develop genetic, biochemical and structural methods to discover and characterize bilayer-bound machines in molecular detail and to learn how they function within intricate cellular pathways. We build genome-scale maps of genetic interaction networks in model organisms to identify multi-component complexes and to infer their functions. We develop methods for reconstituting membrane-binding proteins in the presence of model membranes that mimic the in vivo target membrane in topology, lipid composition, size, and shape. We then leverage advances in cryo-electron microscopy (cryoEM) with our novel image analysis algorithms to solve structures of these machines in their native, membrane-associated states. These innovative tools are distinct methodologically but reinforcing: our genetic maps first define complexes for in depth in vitro study; and after reconstitute and solve 3D reconstructions by cryoEM we probe the functional predictions of our models with loss-of-function versus second-site suppression assays in vivo. These orthogonal approaches thus provide synergistic evidence for the mechanisms that drive cellular functions and result in genuine atomic-resolution understanding of the structures and functions we study. In concert with technology advances in electron optics and electron detectors, my laboratory is overcoming the genetic, biochemical and computational challenges responsible for the lack of structural models for membrane-bound protein assemblies. The aims outlined here are first, critical steps in our long-term research program to determine how the mechanisms we discover are corrupted by disease or hijacked by pathogens. Our ultimate objective is to translate this understanding into effective and tolerable treatments for diseases caused by defective or co-opted membrane-associated machines.

描述(由申请人提供)：本提案的统一目标是了解在细胞膜上组装的机器的结构和功能。细胞依靠这些机器来改变其膜的形状、大小和连通性，这种“重塑”是细胞分裂、迁移、分化和交流的基础。此外，每个病原体都劫持或破坏与膜相关的复合体，以感染细胞或逃离细胞。尽管有如此重要的意义，但我们缺乏关于膜结合蛋白如何传递信号、寡聚或重塑细胞膜的大小、形状和拓扑结构的全面模型。为了克服在研究瞬时组装在膜上的多组分复合体方面的内在挑战，我们将开发遗传学、生化和结构方法来发现和表征分子细节上的双层结合机器，并了解它们如何在复杂的细胞通路中发挥作用。我们在模型生物体中建立基因组规模的遗传相互作用网络图，以识别多组分复合体并推断它们的功能。我们开发了在模型膜存在的情况下重建膜结合蛋白的方法，该模型膜在拓扑结构、脂类组成、大小和形状上模仿体内的靶膜。然后，我们利用冷冻电子显微镜(CryoEM)的进步和我们的新图像分析算法来解决这些机器在其固有的膜相关状态下的结构。这些创新的工具在方法上是不同的，但却是强化的：我们的基因图谱首先定义了用于深入体外研究的复合体；在通过CryoEM重建和解决3D重建之后，我们在体内通过功能丧失和第二位点抑制分析来探索我们模型的功能预测。因此，这些正交方法为驱动细胞功能的机制提供了协同证据，并导致对我们研究的结构和功能的真正原子分辨率理解。随着电子光学和电子探测器技术的进步，我的实验室正在克服遗传、生化和计算方面的挑战，这些挑战是导致缺乏膜结合蛋白质组装的结构模型的原因。这里概述的目标是我们长期研究计划中的第一个关键步骤，以确定我们发现的机制是如何被疾病破坏或被病原体劫持的。我们的最终目标是将这一理解转化为有效和可耐受的治疗方法，用于治疗由缺陷或增选的膜相关机器引起的疾病。