Toward Atomic Resolution of Membranes and Membrane-Associated Machines

描述（由申请人提供）：本提案的统一目标是了解组装在细胞膜上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，每种病原体都会劫持或破坏膜相关复合物以感染或逃离细胞。尽管如此重要，但我们缺乏关于膜结合蛋白如何转导信号、寡聚或重塑细胞膜大小、形状和拓扑结构的综合模型。为了克服研究在膜上瞬时组装的多组分复合物的内在挑战，我们将开发遗传，生化和结构方法来发现和表征双层结合机器的分子细节，并了解它们如何在复杂的细胞途径中起作用。我们在模式生物中建立基因相互作用网络的基因组尺度图，以识别多组分复合物并推断其功能。我们开发了在模型膜存在的情况下重组膜结合蛋白的方法，这些模型膜在拓扑结构、脂质组成、大小和形状上模仿体内靶膜。然后，我们利用冷冻电子显微镜（cryoEM）的进步和我们新颖的图像分析算法来解决这些机器在其原生膜相关状态下的结构。这些创新的工具在方法上是独特的，但加强：我们的基因图谱首先定义了深入体外研究的复合物；在通过冷冻电镜重建和解决三维重建后，我们通过功能丧失与体内第二位点抑制实验来探索我们的模型的功能预测。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，这些挑战是导致膜结合蛋白组装缺乏结构模型的原因。这里概述的目标首先是我们长期研究计划的关键步骤，以确定我们发现的机制如何被疾病破坏或被病原体劫持。我们的最终目标是将这种理解转化为有效和可耐受的治疗方法，以治疗由缺陷或合用膜相关机器引起的疾病。

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.