Silicon nanowires for probing effects of membrane voltage on the TCR-CD3 complex

摘要 T细胞是适应性免疫反应的核心组成部分。为了保护宿主免受各种 不同的病原体，这些细胞必须通过外源肽抗原与其受体的结合而被激活， 在主要组织相容性复合体蛋白（MHC）的背景下，T细胞受体（TCR）。先前 研究已经证明人白血病T细胞的去极化抑制TCR刺激- 通过细胞中的非电压门控Ca 2+通道诱导钙信号传导，这是T细胞的典型标志物 activation.然而，对膜去极化如何调节激活的基本理解 T细胞的状态尚不清楚。由于TCR-CD 3复合物的大小及其在膜中的位置，它具有 获得整个复合体的结构信息一直是一个挑战。据了解， 跨膜（TM）区的残基和胞质结构域中的多碱基残基可能发挥作用， 在复合物组装和调节免疫调节酪氨酸对激酶磷酸化的利用率中的作用 在细胞质中，分别。共同发起人Bezanilla以前的工作已经说明了充电的能力 TM蛋白中的残基在膜电压变化时移动，这对细胞 功能该提案旨在解决的主要问题是T细胞膜的去极化如何能够 调节TCR-CD 3复合物的构象和功能。我们将使用两个新颖的独立硅 申请人最近在Tian实验室开发的纳米材料， T细胞的膜。然后，我们将进行福斯特共振能量转移成像，以检查CD 3 细胞质亚基相对于膜移动以及TCR-CD 3 TM亚基如何相对于TCR移动 细胞外结构域，在纳米材料诱导的去极化和刺激与各种纯化的肽- 亚当斯实验室生产的MHC配体。我们还将研究表达的变化， 在亚当斯实验室中， 膜去极化和TCR刺激。这项工作的结果将提供生物物理和功能 了解质膜电位如何调节T细胞活化， 自身免疫性疾病的治疗策略。

Abstract T cells are central components of the adaptive immune response. In order to protect the host from a variety of different pathogens, these cells must be activated by the binding of foreign peptide antigens to their receptors, the T cell receptors (TCRs), in the context of the major histocompatibility complex protein (MHC). Previous studies have demonstrated that the depolarization of human leukemic T cells dampens TCR stimulation- induced calcium signaling through non-voltage gated Ca2+ channels in the cell, a canonical marker of T cell activation. However, a fundamental understanding of how membrane depolarization regulates the activation state of a T cell is unclear. Due to the size of the TCR-CD3 complex and its location in the membrane, it has been challenging to obtain structural information about the whole complex. It is known that there are charged residues in the transmembrane (TM) region and polybasic residues in the cytoplasmic domain that might play a role in complex assembly and regulating immunomodulatory tyrosine availability for phosphorylation by kinases in the cytoplasm, respectively. Previous work by co-sponsor Bezanilla has illustrated the ability of charged residues in TM proteins to move upon changes in membrane voltage in a manner that is crucial to cellular function. The main question this proposal aims to address is how depolarization of the T cell membrane can regulate the conformation and function of the TCR-CD3 complex. We will use two novel free-standing silicon nanomaterials that the applicant recently developed in the Tian laboratory to optically depolarize the membranes of T cells. We will then perform Förster resonance energy transfer imaging to examine how CD3 cytoplasmic subunits move relative to the membrane and how TCR-CD3 TM subunits move relative to the TCR extracellular domains, upon nanomaterial induced depolarization and stimulation with various purified peptide- MHC ligands produced in the Adams laboratory. We will also study changes in the expression and phosphorylation status of proximal and downstream T cell signaling proteins in the Adams laboratory upon membrane depolarization and TCR stimulation. The results of this work will provide biophysical and functional insight into how plasma membrane potential can regulate T cell activation with potential implications for therapeutic strategies in autoimmunity diseases.