Quantitative Modeling of Lymphocyte Signaling Pathways

淋巴细胞信号通路的定量建模

基本信息

批准号：
7964770
负责人：
Iain Fraser
金额：
$ 9.16万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7964770
关键词：
Agreement Antibodies Antigens CD4 Positive T Lymphocytes Calibration Cell Shape Cells Cellular biology Characteristics Collaborations Computational Biology Computer Simulation Computer software Confocal Microscopy Data Data Quality Detection Discrimination Dyes Feedback Flow Cytometry Goals Human Resources Image Immune Sera Interleukin-2 Laboratories Ligands Lipid Bilayers Lymphocyte Mammalian Cell Measurement Mediating Methods Modeling Molecular Morphology Mus National Institute of Allergy and Infectious Disease Peptides Phosphorylation Phosphotransferases Play Population Distributions Process Proteins Protocols documentation RNA Interference Receptor Activation Receptor Signaling Reporting Role Route Sampling Scaffolding Protein Signal Pathway Signal Transduction Signaling Protein Source Staining method Stains Stimulus T-Cell Receptor T-Lymphocyte Technology Testing Transcription Factor AP-1 Transgenic Mice Validation Western Blotting Work base cytokine digital extracellular immunological synapse models and simulation numb protein receptor reconstruction response scaffold simulation

项目摘要

Since establishing the Molecular and Cell Biology group in August 2008, lab personnel have been hired and significant progress made in acquiring preliminary data for computational simulations of TCR-mediated Erk activation. We have established protocols in the lab for isolation and purification of CD4+ T cells from transgenic mice (5CC7/Rag2) that express T cells against a defined ligand (PCC). In collaboration with Rajat Varmas laboratory, we have acquired a method for generating bead-based lipid bilayers containing the appropriate peptide-loaded MHC and co-stimulatory molecules to provide a reproducible cell-free stimulus to the 5CC7/Rag2 CD4+ T cells. This is important as activation with intact APCs would complicate measurements of Erk cascade protein concentrations and phosphorylation as the cell lysates would contain a mixture of these proteins from both T cell and APC sources. We have generated protein standards for quantificatation of Raf1, Mek1+2, Erk1+2 and Ksr by expressing the murine cDNAs as tagged fusions in bacterial and/or mammalian cells. The optimal expression and purification method has been identified in each case to give acceptable quantities of pure protein for loading reference samples on quantitative western blots. Multiple commercial antibody sources for these targets have been assessed to identify suitable antisera for both western and flow cytometry measurements. We have also assessed the suitability of the xMAP (multi-analyte profiling) technology developed by Luminex for quantitative assessment of protein concentration and phosphorylation. So far, this has provided high quality data for several components of the cascade. We intend to use all of the above approaches to increase confidence in our estimations of both protein number and degree of protein phosphorylation in response to stimulus. Thus far, the data we have obtained for the Mek and Erk proteins expressed in 5CC7/Rag2 CD4+ T cells by both xMAP and quantitative western blotting have shown good agreement. We are currently working on validation of suitable antisera for flow cytometry detection of the Erk cascade proteins. This is important as it provides single cell data and an indication of the population distribution of protein concentrations and phosphorylation responses. We are also developing shRNAs against each target to permit calibration of the quantitative expression data by comparing cells with and without RNAi-based depletion of each Erk cascade protein. Another key requirement in this project is to acquire an estimation of the morphology and geometric characteristics of the T cell to allow accurate calculations of the concentrations of the signaling proteins in the cell. This has been achieved through staining the 5CC7/Rag2 CD4+ T cells with selective dyes and expression of a panel of subcellular markers that localize to specific regions of the cell. In collaboration with the NIAID RTB imaging core laboratory, we have used confocal microscopy and 3D cell reconstruction software to generate reproducible data on cell shape characteristics and volume.

自2008年8月建立分子和细胞生物学组以来，实验室人员已被录用，并在获取初步数据的计算模拟中取得了重大进展。我们已经在实验室中建立了方案，用于从转基因小鼠（5cc7/rag2）分离和纯化CD4+ T细胞，该小鼠（5cc7/rag2）针对定义的配体（PCC）表达T细胞。与Rajat Varmas实验室合作，我们获得了一种生成基于珠的脂质双层的方法，该脂质双层含有适当的肽负载的MHC和共刺激分子，以向5CC7/RAG2 CD4+ T细胞提供可重现的无细胞刺激。这很重要，因为用完整的APC激活会使ERK级联蛋白浓度和磷酸化的测量复杂化，因为细胞裂解物将包含来自T细胞和APC来源的这些蛋白质的混合物。我们通过将鼠cDNA表示为细菌和/或哺乳动物细胞中标记的融合量来定量RAF1，MEK1+2，ERK1+2和KSR的蛋白质标准。在每种情况下，已经确定了最佳表达和纯化方法，以提供可接受的纯蛋白质，用于在定量的蛋白质印迹上加载参考样品。已经评估了针对这些靶标的多种商业抗体来源，以鉴定西方和流式细胞仪测量的合适抗血清。我们还评估了Luminex开发的XMAP（多分析物分析）技术的适用性，用于定量评估蛋白质浓度和磷酸化。到目前为止，这为级联的几个组件提供了高质量的数据。我们打算使用上述所有方法来提高对蛋白质数量和蛋白质磷酸化程度的估计，以响应刺激。到目前为止，我们为通过XMAP和定量Western blotting在5cc7/rag2 CD4+ T细胞中表达的MEK和ERK蛋白获得的数据表现出了很好的一致性。我们目前正在验证合适的抗血清用于流式细胞仪检测ERK级联蛋白。这很重要，因为它提供了单细胞数据以及蛋白质浓度和磷酸化反应的种群分布的指示。我们还针对每个目标开发了shRNA，以通过比较有或没有基于RNAi的每个ERK级联蛋白的细胞来校准定量表达数据。该项目的另一个关键要求是获取T细胞的形态和几何特征的估计，以准确计算细胞中信号蛋白的浓度。通过用选择性染料和一组定位于细胞特定区域的亚细胞标记物的表达染色5cc7/rag2 cd4+ T细胞来实现这一目标。与NIAID RTB成像核心实验室合作，我们使用了共聚焦显微镜和3D细胞重建软件来生成有关细胞形状特性和体积的可重复数据。