Microproteomic analysis of laser capture microdissected cells forming TNTs

描述(申请人提供)：在这个项目中，我们打算通过探索激光捕获显微解剖(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(TNTs)的小薄膜突起。我们的研究集中在TNTs的特征上，TNTs是一种新的细胞间功能连接机制，在病毒、导致神经退行性疾病的错误折叠的蛋白质聚集体和癌症的传播中。TNTs存在于多种细胞类型中，可以运输细胞质和膜结合的分子、细胞器和传播病原体。在体外，这些结构是异质的，在结构和功能上都出现了许多差异。体内和组织外植体中也存在类似的结构。遗憾的是，S目前对TNT形成的基本机制、结构成分、运输机制或信号转导途径知之甚少。传统的蛋白质组学技术诱人地提供了帮助阐明许多这些悬而未决的问题的潜力。但研究TNTs的研究人员在使用传统技术时往往面临两个重大障碍。第一个是下游MS分析所需的大量细胞(通常约为10,000个细胞)。第二个是获得显著结果所必需的高度样本均匀度。事实上，与TNTs合作的困难在于这些TNTs的暂时性 结构。在所有细胞中，细胞并不总是形成连接。因为过去的研究人员 在克服这些障碍方面找不到任何帮助，对TNTs的研究已经沦为“钓鱼”，寻找潜在的重要蛋白质，减缓了该领域的进展。幸运的是，最近的研究表明，TNTs可以由蛋白质过度表达或在应激条件下诱导，并为克服TNTs的暂时性提供了希望。我们发现了一种名为Myo10的蛋白质，与对照细胞相比，它可以使被TNTs连接的细胞的相对百分比增加50%以上。这种可重复的TNT诱导方式最终为我们提供了一个工具来阐明TNT功能和形成所需的蛋白质。在这里，我们计划使用该蛋白来诱导TNT的形成，然后使用LCM进一步丰富样本群体。我们将使用MALDI-MS等微蛋白质组学技术分析这些丰富的样本，这将允许检测和比较实验条件之间的蛋白质表达差异。此外，与需要10-50克蛋白质的传统蛋白质组学技术不同，微蛋白质组学只需要1-2克蛋白质就可以进行分析。总体而言，虽然这个项目在技术上具有挑战性，需要耐心和故障排除，以确定拟议分析的最佳条件，但我们相信，将这些新技术结合起来并开拓这一领域是一个很好的机会。此外，更好地表征TNTs的形成和功能将提供重要的见解，并导致新的靶点，以改善神经保护，以抵御Pron或其他蛋白质病，以及对抗HIV-1感染和癌症。

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 ?g of protein, microproteomics requires only 1-2 ?g 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.