Decoupling Receptor Clusters and Signaling Crosstalk in Phagosome Membranes

项目摘要/摘要 免疫细胞中的大内体，称为吞噬小体，含有对病原体具有不同功能的受体。 摄取和炎症反应。这些受体协同作用，以最大限度地提高免疫反应。 感染性病原体；它们的错误合作与许多慢性炎症性疾病有关。据推测， 受体形成簇以促进它们的协同功能。然而，目前还没有直接证据表明， 支持这一猜测。关键的障碍是缺乏方法来直接衡量 吞噬小体上的受体簇，因为吞噬小体是细胞内的间隔，不能直接 一旦形成，就可以从外部进入。该项目的长期目标是通过以下方式阐明机制 哪种吞噬小体受体聚集并协同调节整体免疫反应。朝向那个方向 目标，这项R03试点研究的重点是了解Dectin-1和Dectin-1之间的集群和信号串扰 巨噬细胞中的Toll样受体2(TLR2)。这项提议的目标是获得直接的试点证据，以 验证长期以来的假设，即Dectin-1和TLR2聚集在吞噬体膜上进行交叉调节 免疫反应。我们将首先建立一种方法，在物理上分离受体簇和它们的 吞噬体膜上的信号串扰。而不是通常用作 吞噬靶，我们将使用具有两个不同半球的粒子(称为Janus粒子)，每个粒子具有 特定受体所特有的配体。一旦被包裹在吞噬小体内， 颗粒上的两个配体将迫使相应的受体和信号蛋白在没有 改变吞噬体膜的整体化学成分。细胞反应产生的变化 通过这一操作将揭示受体聚集的功能。这种空间解耦的可行性 战略已经在我们的初步结果中得到了展示。在目标1中，我们将使用这种方法来确定 Dectin-1和TLR2招募适配蛋白CARD9以实现信号串扰的机制。 在目标2中，我们将确定Dectin-1和TLR2聚集在交叉调节巨噬细胞中的作用 免疫反应，通过控制Dectin-1和TLR2在吞噬小体上的解偶联和聚集。这 R03研究将证明Dectin-1和TLR2聚集之间的因果关系及其 免疫反应的协同调节。它还将建立第一种描绘受体的方法 仅通过物理手段的相互作用和信号传递，与遗传和药理学的不同 本质上是化学性质的方法。因为吞噬小体中的受体串扰对许多人来说都是常见的 不同的受体和不同类型的免疫细胞，这种空间去耦合策略将打开一扇门 这是一个尚未探索的研究领域，可能会对该领域产生广泛影响。此R03提案的试点结果将 这对于我们未来将阐明吞噬体内信号串扰的一般机制的研究至关重要。

Project Summary/ Abstract Large endosomes in immune cells, known as phagosomes, contain receptors of diverse functions for pathogen ingestion and inflammatory responses. These receptors cooperate to maximize immune response against infectious agents; their faulty cooperation is implicated in many chronic inflammatory diseases. It is posited that the receptors form clusters to facilitate their synergistic function. However, there is currently no direct evidence supporting this speculation. The critical barrier is the lack of methods to directly measure the functional role of receptor clusters at phagosomes, because phagosomes are intracellular compartments that cannot be directly accessed from the outside once formed. The long-term goal of this project is to elucidate mechanisms by which phagosome receptors cluster and cooperatively regulate the overall immune response. Towards that goal, this R03 pilot study is focused on understanding the clustering and signal crosstalk between Dectin-1 and Toll-like receptor 2 (TLR2) in macrophages. The objective of this proposal is to obtain direct pilot evidence to test the long-standing hypothesis that Dectin-1 and TLR2 cluster at phagosome membranes to cross-regulate the immune response. We will first establish a method that physically decouples receptor clusters and their signaling crosstalk at phagosome membranes. Rather than the homogeneous particles typically used as phagocytic targets, we will use particles with two distinct hemispheres (named Janus particles), each having ligands specific for a particular receptor. Once encapsulated inside phagosomes, the spatial segregation of the two ligands on the particles will force corresponding receptors and signaling proteins to segregate without changing the overall chemical constitution of the phagosome membrane. Changes in cell response produced by this manipulation will reveal the function of receptor clustering. The feasibility of this spatial decoupling strategy has been demonstrated in our preliminary results. In Aim 1, we will employ this approach to identify the mechanism by which Dectin-1 and TLR2 recruit the adaptor protein CARD9 to enable signaling crosstalk. In Aim 2, we will determine the role of Dectin-1 and TLR2 clustering in cross-regulating the macrophage immune responses, by controlling the decoupling and clustering of Dectin-1 and TLR2 on phagosomes. This R03 study will demonstrate the causal relationship between clustering of Dectin-1 and TLR2 and their cooperative regulation of immune response. It will also establish the first method that delineates receptor interactions and signaling through exclusively physical means, in contrast to genetic and pharmacological approaches that are chemical in nature. Because receptor crosstalk in phagosomes is common to many different receptors and various types of immune cells, this spatial decoupling strategy will open the door to an unexplored research area and likely make a broad impact on the field. Pilot results from this R03 proposal will be critical for our future studies that will elucidate the general mechanism of signaling crosstalk in phagosomes.