The role of LAT protein condensation phase transitions in T cell signaling

摘要-项目3 健康的免疫系统依赖于T细胞在接近单一的状态下检测外来激动剂pMHC分子的能力 分子水平，在大量有时非常相似的自身抗原中。当T细胞正常的时候 辨别能力在实验上得到了很好的确立，理解了 TCR信令系统实现了这一点，这远远不是透明的。噪声、变化和 信号保真度是一个严峻的挑战--无论是对于理解T细胞如何工作，还是对于 开发利用T细胞的治疗策略。最近，一类被称为蛋白质的现象 凝聚相变已经开始在生物学中出现。最初是在核背景下确定的 组织和基因表达，在细胞膜上有明显的二维蛋白质凝聚 现已发现在T细胞受体(TCR)信号系统中涉及支架蛋白LAT。而当 LAT在T细胞中作为聚集下游信号分子的支架的作用一直以来都是 公认的实验认识到，这种结构可以通过不同类型的相变形成 过程是较新的。蛋白质缩合相变可以表现出广泛的性质， 与更线性的分子聚集过程有很大不同，因此提供了各种不同的方法来 调节分子信号系统的功能输出。项目3解决了总体上的 假设LAT蛋白质缩合相变的独特性质使 T细胞在抗原识别过程中表现出显著的敏感性和选择性。我们提出了一系列 调查以检验这一假说的各个方面，这些方面结合了重组后的高度定量的实验 在原代T细胞中进行精确的单分子活细胞实验的分子系统。我们有初步的 数据表明，活T细胞中的LAT缩合受一种不寻常的动力学相变控制 而近端TCR信号的特定分子特征被调整以利用这一点。确认 对这一发现的澄清将为我们更详细地调查 TCR信号的相变。而项目1和2侧重于近端信号反馈机制 导致LAT磷酸化和凝析油成核，项目4检查下游信号 从LAT凝析油，项目3强调LAT的实验和计算表征 凝聚相变本身及其性质如何调制下行信号的测量 通过以下具体目标进行传播：1.定义控制LAT启动的因素 T细胞中的凝聚；2.设计非凝聚的LAT和类LAT系统；3.定义LAT如何 凝结物控制RAS和钙离子通路的下游信号传导。从这部作品中获得的见解 将可能解开T细胞受体信号通路机制功能的一些概念谜团 并突出不同的角度来设计和操纵T细胞以获得治疗效益。

ABSTRACT - Project 3 A healthy immune system depends on T cells’ abilities to detect foreign agonist pMHC molecules, at near single molecule levels, among vast numbers of sometimes very similar self-antigens. While the T cell’s fine discrimination capabilities are experimentally well established, understanding how the molecular machinery of the TCR signaling system achieves this is far from transparent. Fundamental issues of noise, variation, and signal fidelity present serious challenges—both for understanding how the T cell works as well as for development of therapeutic strategies utilizing T cells. Recently, a class of phenomena known as protein condensation phase transitions have begun to emerge in biology. Originally identified in the context of nuclear organization and gene expression, a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT. While the role of LAT as a scaffold for the clustering of downstream signaling molecules in T cells has long been recognized, experimental realization that this structure can form through distinct types of phase transition processes is more recent. Protein condensation phase transitions can exhibit a wide range of properties that differ substantially from more linear molecular clustering processes, and thus offer a variety of different ways to regulate the functional output of molecular signaling systems. Project 3 addresses the overarching hypothesis that unique properties of the LAT protein condensation phase transition enable the remarkable sensitivity and selectivity T cells exhibit during antigen recognition. We propose a series of investigations to test aspects of this hypothesis that combine highly quantitative experiments in reconstituted molecular systems with precision single-molecule live cell experiments in primary T cells. We have preliminary data indicating that LAT condensation in live T cells is controlled by an unusual type of kinetic phase transition and that specific molecular features of proximal TCR signaling are tuned to take advantage of this. Confirmation and elucidation of this discovery will form the foundation of our more detailed investigation into the role of the phase transition in TCR signaling. While Projects 1 and 2 focus on proximal signaling feedback mechanisms leading up to LAT phosphorylation and condensate nucleation, and Project 4 examines signaling downstream from the LAT condensate, Project 3 emphasizes experimental and computational characterization of the LAT condensation phase transition itself and measurements of how its properties modulate downstream signal propagation through the following specific aims: 1. Define the factors controlling initiation of LAT condensation in T cells; 2. Engineer non-condensing LAT and LAT-like systems; 3. Define how LAT condensates control downstream signaling to Ras and Ca2+ pathways. Insights originating from this work will likely resolve some conceptual mysteries on mechanistic function of the T cell receptor signaling pathway and highlight alternative angles to engineer and manipulate T cells for therapeutic benefit.