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Technologies to Define and Map Novel Interorganelle Macromolecular Interactions

定义和绘制新型细胞器间大分子相互作用的技术

基本信息

批准号：
9059730
负责人：
NATALIE G. AHN
金额：
$ 39.87万
依托单位：
UNIVERSITY OF COLORADO
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：
2013-07-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9059730
关键词：
Address Apoptosis Binding Biochemical Biochemical Genetics Biochemical Process Biochemistry Biosensor Calcium Cell Polarity Cell membrane Cell physiology Cells Cellular biology Charcot-Marie-Tooth Disease Collaborations Color Communication Complex Endoplasmic Reticulum Environment Eukaryotic Cell FarGo Fluorescence Microscopy Fluorescent Probes Fractionation Goals Golgi Apparatus Health Homeostasis Human Image Integral Membrane Protein Interdisciplinary Study Label Life Link Macromolecular Complexes Maps Mass Spectrum Analysis Mediating Membrane Membrane Proteins Methods Microscopy Mitochondria Morphology Movement Multiprotein Complexes Mutation Oranges Organelles Pathway interactions Phospholipids Population Process Production Protein Engineering Proteins Proteomics Reporter Reporting Research Research Personnel Resolution Resources Role Secretory Vesicles Site Students Supraoptic Vertical Ophthalmoplegia System Technology Time base cell growth cell type cellular imaging design endoplasmic reticulum stress genetic approach human disease imaging modality innovation late endosome live cell imaging mitochondrial metabolism new technology novel programs protein biomarkers protein complex protein crosslink protein profiling red fluorescent protein response screening single cell analysis uptake

项目摘要

DESCRIPTION (provided by applicant): Interorganelle interactions are key processes controlling eukaryotic cell function, and dysregulation of these interactions has been implicated in many human diseases. However, relatively little is known about macromolecular complexes that mediate organelle interactions, due to obstacles that have been difficult to overcome. First, many relevant proteins are integral membrane proteins, which are hard to purify and maintain weak but physiologically important binding interactions. Capturing such interactions by conventional biochemical and genetic approaches is technically difficult. Second, simultaneously tracking transient organelle populations and interactions requires the ability to follow real time dynamics in living cells using multi-color fluorescent probes. However, many fluorescent proteins (FPs) used for live imaging are compromised by the oxidizing environment of many organelles, including ER, Golgi, and secretory vesicles. The goal of this proposal is to define protein complexes that define and modulate novel organelle subpopulations, using a combination of new technologies in mass spectrometry and fluorescent protein based probes for live cell imaging. Our Specific Aims are: (1) Identify candidate protein markers of novel organelles and interorganellar protein complexes. We will develop a proteomics strategy to profile proteins within organelle subpopulations that are dynamic and transient, as well as macromolecular complexes that bridge organelles. (2) Develop novel biosensors to track these protein markers in living cells, by time resolved imaging and high resolution microscopy. We will maximize the available colors of the fluorescent protein spectrum for use in multi-color live cell imaging studies, by solving key problems in fluorescent protein reporters caused by organellar environments that restrict their folding and function. (3) Apply these methods to cutting edge problems in cell biology, addressing mechanisms underlying (i) ER stress and Ca2+-mediated organelle remodeling, (ii) Zn2+ homeostasis, and (iii) cell polarity. We will combine technologies developed in Aims 1 and 2 to create a new experimental workflow which integrates mass spectrometry/proteomics, biosensor design, and high resolution fluorescence microscopy, and apply this to relevant problems in collaborator labs in Aim 3. Our proposal establishes a unique, multidisciplinary collaboration between a team of four investigators, who are leading experts in technologies of proteomics/mass spectrometry, protein engineering and biosensor design, and cutting edge methods for high resolution cell imaging. The combined expertise from these investigators gives us a unique opportunity to discover novel organelles and macromolecular complexes involved in interorganelle contacts, and define their cell biology.

描述(申请人提供)：细胞器间相互作用是控制真核细胞功能的关键过程，这些相互作用的失调与许多人类疾病有关。然而，由于存在难以克服的障碍，人们对调节细胞器相互作用的大分子复合体知之甚少。首先，许多相关蛋白是完整的膜蛋白，它们很难纯化，很难保持微弱的但具有生理意义的结合作用。通过传统的生化和遗传方法捕捉这种相互作用在技术上是困难的。其次，同时跟踪瞬时细胞器种群和相互作用需要使用多色荧光探针跟踪活细胞的实时动态的能力。然而，许多用于实时成像的荧光蛋白(FP)会受到许多细胞器的氧化环境的影响，包括内质网、高尔基体和分泌小泡。这项提议的目标是定义定义和调节新的细胞器亚群的蛋白质复合体，使用质谱学中的新技术和基于荧光蛋白的探针进行活细胞成像。我们的具体目标是：(1)寻找新细胞器和细胞器间蛋白质复合体的候选蛋白质标记。我们将开发一种蛋白质组学策略，以分析细胞器亚群中动态和瞬时的蛋白质，以及连接细胞器的大分子复合体。(2)开发新型生物传感器，通过时间分辨成像和高分辨率显微镜跟踪活细胞中的这些蛋白质标志物。我们将通过解决由于细胞器环境限制其折叠和功能而导致的荧光蛋白质报告中的关键问题，最大限度地利用荧光蛋白质光谱的可用颜色，用于多色活细胞成像研究。(3)将这些方法应用于细胞生物学的前沿问题，解决(I)内质网应激和钙离子介导的细胞器重塑、(Ii)锌离子稳态和(Iii)细胞极性的潜在机制。我们将结合AIMS 1和AIMS 2中开发的技术来创建一个新的实验工作流程，它集成了质谱学/蛋白质组学、生物传感器设计和高分辨率荧光显微镜，并将其应用于AIMS 3中合作实验室的相关问题。我们的提案建立了一个由四名研究人员组成的团队之间独特的多学科合作，他们是蛋白质组学/质谱学、蛋白质工程和生物传感器设计技术以及高分辨率细胞成像尖端方法方面的领先专家。来自这些研究人员的综合专业知识为我们提供了一个独特的机会来发现涉及细胞器间接触的新型细胞器和大分子复合体，并确定它们的细胞生物学。