NER: Dynamic Nanomechanical Mapping of Living Cell Surface Receptors

活细胞必须相互沟通，与环境沟通，并做出适当的反应。这种交流通常由细胞表面受体（CSRs）介导，这些受体以信号分子的形式接收信息，这些信号分子与受体（配体）结合，并通过信号转导级联对该信息作出反应，从而导致细胞反应。因此，csr提供了单元与其环境之间的物理/功能接口。众所周知，细胞的表型和功能与细胞表面的变化有关，包括csr的动态表达和/或分布。然而，目前追踪这些潜在目标和细胞功能信号的能力受到生物复杂性和缺乏实验工具的限制。事实上，目前还没有一种方法可以在异质群体中的单细胞水平上量化细胞状态的这一关键特征，作为对刺激的实时反应的函数，并具有分子分辨率。这种限制是越来越频繁地使用组合方法，高通量筛选说明。寻找细胞类型，生化因子，和合成基质，导致所需的细胞反应。从生物工程的角度来看，如果我们有能力在CSR存在、分布和功能的背景下观察动态的细胞-细胞和细胞-基质相互作用，那么我们可以做很多事情来造福社会（例如，组织和靶向药物递送工程）。从基本的角度来看，关于细胞表面受体的组织和功能，以及它们如何影响信号转导机制，有许多重要但尚未解决的问题，如果我们有能力同时，实时地，用分子分辨率观察这些问题，就可以解决这些问题。VanVliet博士建议，这些知识和工程细胞功能的共同目标将通过对活细胞的CSR景观进行生物功能化的纳米机械成像来实现。这种方法，如果成功的话，将使活细胞表面分子谱的实时变化成为可能，作为遗传、生化和机械刺激的系统变化的功能。VanVliet博士之前通过对无机晶体纳米力学不稳定性的合作研究证明，材料内部的动态结构适应可以通过原子/分子长度尺度上表面的接触变形来测量、诱导和预测。在这个纳米级探索性研究项目中，这些强大的功能将用于动态活细胞表面，通过纳米机械接触，通过定量、同步的结构和生化成像，扩展对CSR界面的基本理解。目标是利用扫描探针显微镜和分子力光谱的潜力，通过化学功能化机械探针与活细胞表面之间的直接、可逆结合，开发一种强大的新型体外细胞表面成像方法。这种方法被称为功能化力成像（FFI），将量化csr和内部但近表面的细胞骨架结构的实时时空分布。与现有的CSR分析方法相比，FFI具有几个明显的优势：这种基于接触的成像不需要从细胞基质中切割细胞，不局限于分析纯化抗体可用的蛋白质，并且可以在单个细胞中随时间跟踪显示CSR的分布和相对结合亲和力。该项目侧重于两个特定方面的发展，这两个方面对于在活细胞中进行纳米力学CSR制图的可行性至关重要：(1)快速、有空间偏差的获取分子间力和结构/机械表面轮廓；(2)在模型细胞系统中验证受体结合特异性，通过互补的实验技术可以控制和量化每个细胞中csr的数量。这些目标将使PI成为可能。其长期目标包括识别间充质干细胞和祖细胞特异性CSR特征，然后通过外部机械刺激跟踪和指导这些细胞的分化，应用范围从组织工程血管到化学机械传感器。这项研究的智力价值将包括一种新的、定量严谨的实验工具，它将催化生物系统中化学-力学耦合的创新、跨学科研究。该项目的更广泛影响包括一门新的基于实验室的纳米力学生物实验课程，该课程将提供给麻省理工学院、韦尔斯利学院和布兰代斯大学的本科生，该课程将由参与上述研究目标的研究生研究助理共同指导。本提案解决了NSF NES征集的纳米尺度生物系统研究和教育主题。

Living cells must communicate with each other and with their environment and respond appropriately. This communication is generally mediated by cell surface receptors (CSRs) which receive information in the form of signalling molecules that bind to the receptors (ligands)and respond to that infomation via a signal transduction cascade that leads to a cellular response. Thus, CSRs provide the physical/functional interface between the cell and its environment. It is known that cell phenotype and function are correlative with changes in the cell surface, including the dynamic expression and/or distribution of CSRs. However, the present capacity to track these potential targets and signals of cell function is limited by both biological complexity and a lack of experimental tools. In fact, there exists no current method to quantify this critical signature of cell state at the level of single cells within heterogeneous populations, as a function of real-time response to stimuli, and with molecular resolution. This constraint is illustrated by the increasingly frequent use of combinatorial approaches, high-throughput screening.to find cell types, biochemical factors, and synthetic substrata that result in the desired cell response. From a bioengineering perspective, there is much that could be done to benefit society (e.g., tissue and targeted drug delivery engineering) if we had the capacity to observe dynamic cell-cell and cell-substrate interactions in the context of CSR presence, distribution, and function. From a basic fundamental perspective, there are many important but as-yet-unanswerable questions about cell surface receptor organization and function and how these effect signal transduction mechanisms that could be addressed if we had the capacity to observe these things simultaneously, in real time, with molecular resolution. Dr. VanVliet suggests that such knowledge and the concurrent goal of engineered cell function will be enabled via biofunctionalized nanomechanical imaging of the CSR landscape on living cells. This approach, if successful, will enable unparalleled access to real-time changes in the molecular profile of living cell surfaces, as a function of systematic variation of genetic, biochemical and mechanical stimuli. Dr. VanVliet has previously demonstrated, via collaborative investigations of nanomechanical instabilities in inorganic crystals, that dynamic structural adaptations within materials can be measured, induced, and predicted via contact deformation of surfaces at atomistic/molecular length scales. In this Nanoscale Exploratory Research project, these powerful capabilities will be developed for dynamic living cell surfaces, in order to extend fundamental understanding of the CSR interface through quantitative, simultaneous structural and biochemical imaging via nanomechanical contact. The objective is to capitalize on the potential of scanning probe microscopy and molecular force spectroscopy to develop a powerful new in vitro cell surface imaging approach through direct, reversible binding between a chemically functionalized mechanical probe and the living cell surface. This approach, termed functionalized force imaging (FFI), will quantify the real-time spatial and temporal distribution of CSRs and internal but near-surface cytoskeletal structure. FFI presents several distinct advantages over existing approaches for CSR analysis: This contact-based imaging does not require cleavage of the cell from its substratum, is not restricted to the analysis of proteins for which purified antibodies are available, and reveals the distribution and relative binding affinity of CSRs that can be tracked over time within individual cells. This project focuses on the development of two specific aspects critical to the feasibility of nanomechanical CSR mapping in living cells: (1) rapid, spatially biased acquisition of both intermolecular forces and structural/mechanical surface profiles; (2) validation of receptor binding specificity in a model cell system for which the number of CSRs per cell can be controlled and quantified through complementary experimental techniques. These objectives will enable the PI.s longer-term goals which include identifying mesenchymal stem and progenitor cell-specific CSR profiles, and then tracking and guiding the differentiation of these cells through external mechanical stimuli for applications ranging from tissue engineered vasculature to chemomechanical sensors. The intellectual merit of the research will include a new, quantitatively rigorous experimental tool that will catalyze innovative, cross-disciplinary investigations of chemomechanical coupling in biological systems. This broader impact of this project includes a new laboratory-based course on nanomechanical biological experimentation that will be offered to undergraduates of MIT, Wellesley College, and Brandeis University, and that will be co-instructed by the graduate research assistant participating in the above research objectives. This proposal addresses the Biosystems at the Nanoscale research and education theme of the NSF NES solicitation.