Quantitative Modeling of Lymphocyte Signaling Pathways

自2008年8月成立分子和细胞生物学小组以来，已经聘请了实验室人员，并在获取TCR介导的ERK激活的计算模拟的初步数据方面取得了重大进展。 我们已经在实验室建立了从转基因小鼠(5CC7/Rag2)中分离和纯化CD4+T细胞的方案，这些转基因小鼠表达针对特定配体(PCC)的T细胞。在与Rajat Varmas实验室的合作中，我们获得了一种产生基于珠粒的脂质双层的方法，该方法包含适当的多肽负载MHC和共刺激分子，为5CC7/Rag2 CD4+T细胞提供可重复的无细胞刺激。这一点很重要，因为激活完整的APC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。 我们通过在细菌和/或哺乳动物细胞中以标记融合的形式表达小鼠的cDNA，建立了Raf1、MEK1+2、ERK1+2和KSR的定量蛋白质标准。在每种情况下都确定了最佳的表达和纯化方法，以获得可接受的纯蛋白质数量，用于定量Western blotting上的参考样本。已经对这些靶标的多种商业抗体来源进行了评估，以确定适合于免疫和流式细胞术测量的抗血清。我们还评估了Luminex开发的xMAP(多分析物分析)技术对蛋白质浓度和磷酸化的定量评估的适用性。到目前为止，这为级联的几个组成部分提供了高质量的数据。我们打算使用所有上述方法来增加我们对蛋白质数量和对刺激反应的蛋白质磷酸化程度的估计的信心。到目前为止，我们通过xMAP和定量Western blotting获得的在5CC7/Rag2 CD4+T细胞中表达的MEK和Erk蛋白的数据都显示出很好的一致性。我们目前正致力于验证合适的抗血清，用于流式细胞术检测ERK级联蛋白。这一点很重要，因为它提供了单细胞数据，并指示了蛋白质浓度和磷酸化反应的总体分布。我们还针对每个靶点开发shRNAs，通过比较有没有基于RNAi的ERK级联蛋白缺失的细胞来校准定量表达数据。 这个项目的另一个关键要求是获得T细胞的形态和几何特征的估计，以便准确计算细胞中信号蛋白的浓度。这是通过用选择性染料对5CC7/Rag2 CD4+T细胞进行染色并表达一组定位于细胞特定区域的亚细胞标记来实现的。在与NIAID RTB成像核心实验室的合作下，我们使用共聚焦显微镜和3D细胞重建软件来生成关于细胞形状特征和体积的可重复数据。

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.