Measuring the effect of mechanical forces on receptor signals through nanoscale AFM measurement of the cytoskeleton

主要研究者：Manish J. Butte提案ID：1264833本提案的研究目的是检验细胞骨架上的机械力调节来自细胞表面受体的信号的假设。在T细胞中，已经表明除了T细胞受体的连接之外，外源性机械力似乎是T细胞被激活所必需的。在癌细胞中，已知机械力加上细胞外基质对整联蛋白受体的连接可导致侵袭性和增殖性表型。力是如何作为改变分子信号事件的线索的？最近在我的实验室使用生物原子力显微镜（AFM）的发现表明，幼稚T细胞具有僵硬的细胞骨架结构，在TCR触发后立即软化，并且细胞骨架软化的设施，由激酶和拴系分子介导，实际上控制激活阈值。这项建议旨在测试细胞骨架结构调节细胞表面受体信号的能力，从而将机械生物学和信号传导领域联系在一起。虽然细胞骨架在细胞运动和细胞内运输中的重要作用已得到很好的表征，但其在受体信号传导过程中的功能作用在很大程度上尚未被探索。PI实验室的研究结果促使我们在细胞水平上研究机械力的作用，研究持久的细胞骨架结构如何控制细胞的激活或侵袭，并在单个受体水平上研究力如何调节受体信号。该项目的智力价值在于PI控制和研究纳米级力与受体信号之间联系的独特能力。能够连接活细胞上的受体，并通过AFM传递力，再加上通过旋转圆盘共聚焦显微镜同时成像，是我实验室独特而强大的能力。将会出现的是对细胞如何感知、施加和“存储”力信号的更好理解。从PI在T细胞中的发现推断，通常，细胞骨架系链的松动控制受体信号传导的阈值。细胞可以将机械力解释为门控受体信号作为防止无意触发的一种方式（即，自身免疫），或作为利用在某些发育、胚胎检查点期间发生的细胞间力作为增殖和生长的触发的一种方式。以细胞骨架结构的形式“储存”这些受体设定点可能比例如酪氨酸磷酸化或基因转录更持久。这项工作的更广泛影响包括一个教育推广计划，该计划提出开发工具，通过直接经验教授纳米生物学。PI利用最新的3D打印技术打印手掌大小的细胞模型，并具有纳米级特征。这些细胞模型可以被拿着和触摸来采样细胞特征，并将激发人们对纳米科学和生物学的兴趣。结合这些模型，研究人员正在开发一种触觉界面，允许用户在使用3D操纵杆和AFM时体验“触摸”细胞的纳米级特征。建议与当地教室和科学博物馆共享这些细胞模型和触觉界面，以激发所有年龄段的参与者与细胞的纳米级特征和动态运动进行互动。除了这种教育影响之外，该项目将有助于建立一个具有细胞生物力学专业知识的独特研究小组，丰富有兴趣将通过这项工作开发的方法应用于细胞及其生物学问题的跨学科合作者小组。研究结果将通过出版物和网站传播，使学生和研究人员能够使用AFM技术。最后，PI将与当地AFM初创公司Molecular Vista以及Agilent Technologies合作，将机械生物学见解构建到他们的AFM仪器中。该提案解决了美国国家工程院的重大挑战之一：设计生物科学发现的工具。

PI: Manish J. ButteProposal ID: 1264833The research objective of this proposal is to test the hypothesis that mechanical forces upon the cytoskeleton regulate signals from cell-surface receptors. In T cells it has been shown that exogenous mechanical forces in addition to ligation of the T cell receptor seem to be required for the T cell to become activated. In cancer cells, it is known that mechanical forces plus ligation of integrin receptors by the extracellular matrix can lead to an invasive and proliferative phenotype. How does force act as a cue to modify molecular signaling events? Recent discoveries in my lab using biological Atomic Force Microscopy (AFM) showed that naive T cells have a stiff cytoskeletal architecture, which softens immediately upon TCR triggering, and that the facility with which the cytoskeleton softens, mediated by kinases and tethering molecules, actually controls the threshold for activation. This proposal seeks to test generally the capability of cytoskeletal structures to modulate the signals of cell-surface receptors, thus tying together the fields of mechanobiology and signaling. While the cytoskeleton's important roles in cell motility and intracellular trafficking are well characterized, its functional roles during receptor signaling have been largely unexplored. The PI lab's results prompt to investigate the role of mechanical forces at the cellular level, to study how durable cytoskeletal structures control the activation or invasiveness of cells, and at the single receptor level, to show how force upon the receptors modulates its signaling.The Intellectual Merit of this program lies in PI's unique ability to control and study the linkage between nanoscale forces and receptor signaling. The ability to ligate receptors on live cells and deliver forces by AFM, plus image simultaneously by spinning disk confocal microscopy, is a unique and powerful capability of my lab. What will emerge will be an improved understanding of how force signals are sensed, exerted, and "stored" by cells. It is inferred from PI's findings in T cells that, generally, loosening of cytoskeletal tethers controls the threshold of receptor signaling. Cells may interpret mechanical forces to gate receptor signals as a way of preventing inadvertent triggering (i.e., autoimmunity), or as a way of utilizing inter-cellular forces that occur during certain developmental, embryonic checkpoints as a trigger for proliferation and growth. "Storing" these receptor set-points in the form of cytoskeletal structures may be more durable than, for example, tyrosine phosphorylation or gene transcription.The Broader Impact of this work includes an Educational Outreach Plan that proposes to develop tools to teach Nanoscale Biology by direct experience. The PI has utilized the latest 3D printing technology to print palm-sized cell models complete with nanoscale features. These cell models can be held and touched to sample cellular features, and will inspire interest in nanoscale science and biology. In conjunction with the models, the investigators are developing a haptic interface that allows users to experience "touching" the nanoscale features of cells when using a 3D joystick and the AFM. It is proposed to share these cell models and haptic interfaces with local classrooms and science museums, to inspire participants of all ages to interact with the nanoscale features and dynamic movements of cells. Beyond this educational impact, this project will help build a unique research group with expertise in cellular biomechanics, enriching interdisciplinary group of collaborators who are interested in applying the methods developed through this work to their cells and their biological questions. The findings will be disseminated though publications and the website, making the AFM techniques developed accessible to students and researchers. Finally, the PI will collaborate with Molecular Vista, a local AFM startup, and with Agilent Technologies to build the mechanobiological insights into their AFM instruments. This proposal addresses one of the National Academy of Engineering's Grand Challenges: to engineer the tools of biological scientific discovery.