High Resolution Characterization of Receptor Signaling and Regulation

 描述（由申请人提供）：这项持续的研究旨在以微米和纳米级分辨率表征细胞信号传导的集体分子事件，这些事件由质膜受体启动，并在空间和时间上高度协调。目前的研究重点主要集中在肥大细胞上IgE的高亲和力受体Fc ε RI上，它在肥大细胞中起着重要的作用。 在过敏性免疫应答中起关键作用，并作为受体介导的细胞信号传导的有价值的模型系统。这些研究的主要目的是阐明质膜环境中发生的蛋白质和脂质的结构相互作用，这些相互作用通过IgE-Fc ε RI的抗原交联而改变，并导致细胞内信号级联的跨膜触发，最终导致细胞对刺激物的反应。拟议的研究将探讨肌动蛋白细胞骨架调节这些相互作用在多个层面上的机制，包括膜钉扎和区室化的皮质网络，防止自发启动的信号和重排与细胞粘附到基板，调整反应水平。Specific Aim 1将使用双色超分辨率荧光显微镜成像并分析在IgE-Fc ε RI交联后立即启动的质膜中信号组分之间的动态相互作用，并将通过对膜和细胞骨架的选择性扰动来评价这些信号组分的调节。结构上定义的配体将被用来辨别交联的特征，这些特征对于信号的引发是至关重要的，并且这些实验结果将被纳入信号系统的计算模型中。具体目标2将使用多功能微图案化表面和荧光显微镜研究由成簇的IgE-Fc ε RI引发的后期膜相互作用，从而导致下游信号传导和由整合素结合调节的细胞应答事件。选择性抑制剂和分子张力传感器将被用来探测肌动蛋白细胞骨架和整合素在施加机械力和调节信号网络中的参与，这与单细胞测定中随后的细胞反应有关。具体目标3将转化从IgE-Fc ε RI模型系统的详细表征中获得的生物物理学见解和高分辨率方法，以研究参与神经细胞活动并在神经退行性疾病中被破坏的质膜相互作用。这些高度合作研究的目标是为重大生物医学影响奠定基础并建立富有成效的方向。初始实验 将评估帕金森病中可能发生的α-突触核蛋白的细胞相互作用、与神经元可塑性信号相关的神经营养因子受体p75的细胞表面聚集以及似乎由小胶质细胞形成的溶酶体突触，以水解与阿尔茨海默病相关的A β原纤维。

 DESCRIPTION (provided by applicant): This continuing research is aimed at characterizing, with micro- and nanoscale resolution, the collective molecular events of cellular signaling that are initiated by plasma membrane receptors and are highly orchestrated in space and time. Focus remains primarily on the high affinity receptor for IgE, FcεRI, on mast cells, which plays a pivotal role in the allergic immune response and serves as a valuable model system for receptor-mediated cellular signaling. The primary goal of these studies is to elucidate structural interactions of proteins and lipids occurring within the plasma membrane milieu that are altered by antigen crosslinking of IgE- FcεRI and result in transmembrane triggering of intracellular signaling cascades, culminating in cellular responses to the stimulant. Proposed research will investigate mechanisms by which the actin cytoskeleton regulates these interactions at multiple levels, including membrane pinning and compartmentalization by the cortical meshwork that prevents spontaneous initiation of signaling and rearrangements associated with cell adherence to a substrate that adjust the level of response. Specific Aim 1 will use two-color super resolution fluorescence microscopy to image and analyze dynamic interactions among signaling components in the plasma membrane that are initiated immediately after IgE-FcεRI crosslinking, and regulation of these will be evaluated by selective perturbation to membrane and cytoskeleton. Structurally defined ligands will be employed to discern features of crosslinking that are critical for signal initiation, and these experimental results will be incorporated into computational models of signaling systems. Specific Aim 2 will use versatile micro- patterned surfaces and fluorescence microscopy to investigate later-stage membrane interactions initiated by clustered IgE-FcεRI leading to downstream signaling and cellular response events that are modulated by integrin engagement. Selective inhibitors and molecular tension sensors will be employed to probe participation of the actin cytoskeleton and integrins in imposing mechanical forces and regulating signaling networks, as related to consequent cellular responses in single cell assays. Specific Aim 3 will translate biophysical insight and high-resolution approaches gained from detailed characterization of the IgE-FcεRI model system to investigate plasma membrane interactions that participate in neurological cell activities and are disrupted in neurodegenerative diseases. The goal of these highly collaborative studies is to lay the groundwork and establish fruitful directions for significant biomedical impact. Initial experiments will evaluate cellular interactions of α-synuclein that may occur in Parkinson's disease, cell surface clustering of neurotrophin receptor p75 related to signaling in neuronal plasticity, and lysosomal synapses that appear to be formed by microglia to hydrolyze Aβ fibrils associated with Alzheimer's disease.