EAGER: Dynamic Affinity for Regulation of Cell Signaling

PI：程东建议ID：1330663概述：了解细胞-微环境通信的能力不仅在基础生命科学研究中非常重要，而且在癌症研究和再生医学等各种生物医学应用中也是重要的。由于细胞受体是细胞感知其微环境的主要参与者，因此对细胞-微环境交流的理解需要深入了解细胞受体与其配体之间的分子相互作用，这些配体可以是另一个细胞的细胞受体、游离生物分子或细胞外基质成分。然而，目前对这些生物分子相互作用的理解主要来自于对处于固定或最佳功能状态的细胞受体的性质的检查，尽管细胞受体及其配体具有动态性质。几乎没有研究了解动态亲和力如何驱动细胞间的交流，最终调节细胞内的信号通路。因此，这一提议旨在通过工程和生物方法来探索动态亲和力如何决定细胞内信号传递。智力优势：本提议的目的是发现和理解一种由动态亲和力驱动的细胞信号传递调节的新机制。为了实现这一目标，CD82和整合素将被用作受体模型，肿瘤细胞和内皮细胞将被用作细胞模型。之前的研究，包括在Pi的实验室进行的研究表明，CD82与整合素有关，分子相互作用可能影响细胞表型。然而，由于缺乏进行这些分析的实验技术，整合素结合亲和力是否会改变内皮信号在很大程度上仍不清楚。这些研究支持的假设是，可诱导CD82表达的增加降低了整合素结合的亲和力，从而抑制了肿瘤诱导的缝隙形成和肿瘤细胞的外渗。为了验证这一未经检验的假说，提出了两项高风险任务。第一个任务旨在开发一种新的、创新的准三维显微成像技术，以询问整合素的构象状态或结合亲和力如何随着CD82表达水平的变化而变化。第二个任务是了解整合素结合亲和力的动态变化如何决定细胞内信号通路和肌动蛋白动力学。希望通过对该模型系统的研究，深入了解肿瘤细胞与内皮细胞之间的相互作用。具体地说，它将为整合素结合亲和力在肌动蛋白细胞骨架中影响内皮细胞株、蛋白激酶活性以及内皮细胞连接附近VE-钙粘附素分解随时间的变化提供有价值的见解。广泛影响：教育和外联：PI计划建立一个新的跨学科研究和教育计划，旨在提高不同水平的学生，特别是未被充分代表的少数族裔和残疾人在科学和工程领域的参与度。学生将通过现有的外展计划从宾夕法尼亚州的主要教学机构招募。学生将组成研究团队，开发计算机程序，进行实验室工作，分析实验结果，撰写研究论文，在会议上展示研究结果，并保持结果的网络传播。跨学科和变革性特征：这个高风险和高回报的项目反映了一种新的范式，因为以前的研究只专注于结合亲和力如何调节细胞黏附，而不是分子内细胞信号转导，这是这个渴望项目的重点。将在工程学、生物学、生理学和生物物理学的交界处开辟一个新的跨学科研究方向。重要的是，这一提议的成功具有巨大的潜力，可以改变细胞间通讯的研究方式，并为生物医学应用开发当前/未来的生物工程。因此，拟议的研究可能不被视为“常规”问题或方法，并且具有热切机制的适当性。

PI: Cheng DongProposal ID: 1330663Overview:The ability to understand cell-microenvironment communication is important in not only basic life science research, but also various biomedical applications such as cancer research and regenerative medicine. Because cell receptors are the major players for a cell to sense its microenvironment, the understanding of cell-microenvironment communication needs deep insights into molecular interactions between cell receptors and their ligands that can be cell receptors of another cell, free biomolecules, or extracellular matrix components. However, current understandings of these biomolecular interactions come primarily from the examination of the properties of cell receptors at a fixed or optimal functional state despite the dynamic nature of cell receptors and their ligands. Few if any studies have been carried out to understand how dynamic affinities drive intercellular communication that eventually regulates intracellular signaling pathways. Therefore, this proposal is aimed at exploring how dynamic affinity determines intracellular signaling by using both engineering and biological methods.Intellectual Merit:The objective of this proposal is to discover and understand a new mechanism for the regulation of cellular signaling transmission driven by dynamic affinities. To achieve this objective, CD82 and integrins will be used as a receptor model, and tumor cells and endothelial cells will serve as a cell model. Previous studies including those performed at PI's lab have suggeste that CD82 is associated with integrins that molecular interactions may influence cell phenotypes. However, it remains largely unknown on whether integrin binding affinities alter endothelial signaling due to a lack of experimental techniques to perform these analyses. Supported by these previous studies, it is hypothesized that increase of inducible CD82 expression decreases integrin-binding affinities, which inhibits tumor induced gap formation and tumor cell extravasation. Two high-risk tasks are proposed to validate this untested hypothesis. The first task aims to develop a new and innovative quasi-3D microscopy imaging technique to interrogate how integrin conformational states or binding affinities change with the level of CD82 expression. The second task is to understand how the dynamic change of integrin binding affinities determines intracellular signaling pathways and actin dynamics. It is expected that the study of this model system would lead to deep insights into the interactions between tumor cells and endothelial cells. Specifically, it will provide valuable insights into the mechanisms by which integrin-binding affinity affects endothelial cell strains in the actin cytoskeleton, protein kinase activity, and changes in VE-cadherin disassembly near the endothelial cell junctions over time.Broader Impacts:Education and outreach: The PI plans to build a new interdisciplinary research and education program, which aims to improve the participation of students at different levels, particularly underrepresented minorities and persons with disabilities in science and engineering. Students will be recruited through existing outreach programs from predominantly teaching institutions in Pennsylvania. Students will form research teams to develop computer programs, conduct laboratory work, analyze experimental results, write research papers, present findings in conferences, and maintain a web-based dissemination of results. Interdisciplinary and Transformative Traits: This high risk and high payoff project reflects a new paradigm, as prior studies were only focused on how binding affinities regulate cell adhesion rather than intramolecular cell signaling, which is the focus of this EAGER project. A new interdisciplinary research direction will be opened at the interface of engineering, biology, physiology, and biophysics. Importantly, the success of this proposal holds great potential to transform the ways that cell-cell communication is studied, and current / future bioengineering is developed for biomedical applications. Thus, the proposed research may not be viewed as "regular" questions or approaches, and has an appropriateness of the EAGER mechanism.