Microproteomic analysis of laser capture microdissected cells forming TNTs

激光捕获显微切割细胞形成 TNT 的微蛋白质组学分析

基本信息

批准号：
9069895
负责人：
KARINE GOUSSET
金额：
$ 14万
依托单位：
CALIFORNIA STATE UNIVERSITY FRESNO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9069895
关键词：
Alzheimer&apos s Disease Binding Cell Count Cells Co-Immunoprecipitations Data Detection Development Down-Regulation Fishes Fluorescence Microscopy Funding Opportunities Future Grant HIV HIV-1 Health Hydrogen Peroxide Immunoprecipitation In Vitro Individual Industry Infection Lasers Lead Life Malignant Neoplasms Mass Spectrum Analysis Membrane Mentors Methods Microdissection Molecular Machines Nanotubes Nature Neurodegenerative Disorders Organelles Population Prions Protein Analysis Proteins Proteomics Protocols documentation Research Research Personnel Research Proposals Resolution Role Sample Size Sampling Signal Pathway Signal Transduction Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Stress Structure Techniques Technology Time Tissues Training Tubular formation United States National Institutes of Health Virus Western Blotting Work base cell type improved in vivo insight laser capture microdissection mutant neuroprotection novel overexpression pathogen protein aggregate protein expression sample fixation tool vector control

项目摘要

DESCRIPTION (provided by applicant): With this project we propose to analyze small membrane protrusions know as tunneling nanotubes (TNTs) by exploring the limits of laser capture microdissection (LCM) and microproteomic techniques. Our research is focused on the characterization of TNTs, a novel mechanism for functional connectivity between cells, in the spreading of viruses, misfolded protein aggregates that lead to neurodegenerative diseases, and cancer. TNTs have been found in numerous cell types, allowing the transport of cytosolic and membrane-bound molecules, organelles and the spreading of pathogens. In vitro, these structures are heterogeneous and numerous disparities have emerged both in their structures and functions. Similar structures also exist in vivo and in tissue explants. Unfortunately, little s currently known about the basic mechanism of TNT formation, its structural components, transport mechanism or signaling pathways. Traditional proteomic techniques tantalizingly offer the potential to help elucidate many of these unanswered questions. But researchers studying TNTs have often faced two significant obstacles in using traditional techniques. The first is the substantial amounts of cells that are required for downstream MS analysis (usually on the order of 10,000 cells). The second is the high degree of sample homogeneity necessary to obtain significant results. In fact, the difficultly of working with TNTs is the transient nature of these structures. Cells do not form connections at all times, in all cells. Since researchers in the past have found no help in overcoming these obstacles, research on TNTs has been reduced to "fishing" for potentially significant proteins, slowing progress in the field. Fortunately, recent studies have demonstrated that TNTs can be induced by protein over-expression or under stress conditions, and have offered hope in overcoming the transient nature of TNTs. We identified a protein, Myo10, that increases the relative percentage of cells connected by TNTs by over 50% compared to control cells. This reproducible manner of TNT induction finally offers us a tool to elucidate the proteins necessary for TNT function and formation. Here, we plan to use this protein to induce TNT formation and then further enrich sample populations using LCM. We will analyze these enriched samples using microproteomic techniques such as MALDI-MS which should permit the detection and comparison of protein expression differences between experimental conditions. Also, unlike traditional proteomic technologies, that require 10-50 μg of protein, microproteomics requires only 1-2 μg of protein for analysis. Overall, while this project will be technically challenging and will require patience and troubleshooting to identify the best conditions for the proposed analyses, we believe that there is a great opportunity to combine these new techniques and open up the field. Furthermore, a better characterization of both formation and function of TNTs will provide important insights and lead to novel targets to improve neuroprotection against prion or other proteopathies as well as against HIV-1 infection and cancer.

描述（由申请人提供）：通过这个项目，我们建议通过探索激光捕获显微切割（LCM）和微蛋白质组学技术的局限性来分析被称为隧道纳米管（TNT）的小膜突起。我们的研究重点是 TNT（一种 TNT）的表征。在病毒传播、导致神经退行性疾病和癌症的错误折叠蛋白质聚集体中，细胞间功能连接的新机制已在多种细胞类型中被发现，从而允许运输。在体外，这些结构是异质的，并且它们的结构和功能也存在许多差异，不幸的是，目前在体内和组织外植体中还存在很少的结构。人们对 TNT 形成的基本机制、其结构成分、转运机制或信号通路的了解非常有潜力，但研究 TNT 的研究人员经常面临这些问题。使用传统技术有两个重大障碍：第一是下游 MS 分析需要大量细胞（通常为 10,000 个细胞），第二是获得显着结果所需的样品高度同质性。，使用 TNT 的困难在于这些物质的瞬态性自过去的研究人员以来，细胞并不总是在所有细胞中形成连接。由于没有发现克服这些障碍的帮助，对 TNT 的研究已沦为“钓鱼”潜在重要的蛋白质，从而减缓了该领域的进展。幸运的是，最近的研究表明 TNT 可以通过蛋白质过度表达或在应激条件下诱导。，并为克服 TNT 的短暂性带来了希望，我们发现了一种蛋白质 Myo10，与对照细胞相比，这种可重复的 TNT 诱导方式最终使 TNT 连接的细胞的相对百分比增加了 50% 以上。为我们提供了阐明 TNT 功能和形成所需的蛋白质的工具，我们计划使用该蛋白质诱导 TNT 形成，然后使用 LCM 进一步富集样品群体，我们将使用微蛋白质组学技术（如 MALDI-MS）分析这些富集的样品。此外，与需要 10-50 μg 蛋白质的传统蛋白质组学技术不同，微蛋白质组学仅需要 1-2 μg 蛋白质即可进行分析。总体而言，虽然该项目在技术上具有挑战性，并且需要耐心和故障排除来确定拟议分析的最佳条件，但我们相信有一个很好的机会结合这些新技术并开拓该领域，此外，还可以更好地表征该领域。 TNT 的形成和功能将提供重要的见解，并产生新的靶标，以改善针对朊病毒或其他蛋白质病以及 HIV-1 感染和癌症的神经保护。