Molecular Regulation of Matrix Assembly Mechanics

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。细胞生命的基本方面包括生存、定向、运动、生长和成熟。几十年来，科学家们一直在努力了解这些过程的调控，并可能最终为生物体如何发展和适应环境变化提供见解。细胞周围的微环境构成了细胞外基质，细胞外基质本身是一种复杂的蛋白质混合物，由细胞自身产生和组织。这种基质是指导基本细胞功能的化学和机械信号的来源。这个项目将描述生化调节途径和细胞外基质组装过程中发生的机械事件之间的相互作用。这项研究的动机是通过定量分析产生的新数据，同时测量单个基质纤维的组装和细胞在与基质接触的部位施加的拉力。这是通过在具有确定机械性能的硅柱上镀电池来实现的。细胞在每个柱子的顶部形成单独的附着点。然后细胞拉动柱子，细胞在每个附着点施加的拉力的方向性和量是通过测量柱子的最终运动来确定的。这些研究结果确定了驱动基质纤维组装过程的两个机械过程和两个化学调节系统：1)细胞拉力向细胞中心的渐进运动；2)细胞周围的压缩松弛；3)一种叫做黏附激酶的酶的激活；4)组织细胞骨架的mDia1蛋白失活。该研究项目将推进这些发现，并提供见解，将解决有关在细胞外基质组装过程中聚焦力和形状应变的分子信号过程的基本问题。首席研究员生产了新颖的工具，组建了一个世界级的多学科团队，并利用优秀的资源进行细胞和分子成像和力测量，这将增强这项工作的科学影响。该项目将进一步努力建立一个具有挑战性和支持性的跨学科环境，在这个环境中，不同背景的学生可以培养批判性思维和表达能力，并开发有利于科学界的新工具。迄今为止，这些努力的成果包括：促进和包容不同群体的学生和博士后受训者，包括女性和代表性不足的少数群体，参与假设驱动和发现科学；发起一个互动的联合实验室会议，包括约翰霍普金斯大学生物化学、生物物理学、细胞生物学和工程学的实验室，以便在一个具有多种科学观点的合作论坛上为学生提供建立演示和实验设计技能的机会；研究合作，包括NSF仪器资助，最近为更广泛的霍普金斯社区提供了一种新的，最先进的原子力显微镜；科研伦理教学的创新提供新的图像处理工具，供科学界免费下载，不受限制地使用，这些工具目前正在为美国、法国、印度和澳大利亚的调查人员使用。目前的研究项目将推动这些努力。通过与范安德尔研究所的一个研究小组的新合作，将有机会学习测量信号分子之间相互作用的新技术。通过与杜克大学的基质和细胞生物学实验室以及约翰霍普金斯大学的生物物理学和生物学实验室的互动，该项目将进一步加强跨学科和机构间的合作。该小组的专业知识和资源的广度将优化研究计划的成功，并确保学生广泛接触多个学科。该计划的广度和协同作用将在不同学科之间建立新的联系，并为其他研究人员提供物理学和生物学之间界面的新范例。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual Merit Essential aspects of cellular life include survival, orientation, movement, growth, and maturation. Efforts to understand the regulation of these processes has intrigued scientists for decades, and may ultimately provide insights into how organisms develop and adapt to environmental change. The microenvironment immediately surrounding cells makes up the extracellular matrix, which itself is a complex mixture of proteins that are produced and organized by the cells themselves. This matrix is a source of chemical and mechanical signals that direct essential cellular functions. This project will characterize the interactions between biochemical regulatory pathways and mechanical events that occur during extracellular matrix assembly. This research is motivated by new data generated with a quantitative assay that simultaneously measures the assembly of individual matrix fibers and the pulling force that cells exert at sites of contact with the matrix. This is accomplished by plating cells on groups of silicon posts with defined mechanical properties. Cells form individual attachment sites at the top of each post. Cells then pull on the posts, and the directionality and quantity of pulling force that a cell exerts at each attachment site is determined by measuring the resulting movement of the post. Results from these studies identified two mechanical processes and two chemical regulatory systems that drive the progress of matrix fiber assembly: 1) The progressive movement of cell pulling force toward the cell center; 2) the relaxation of compression in the periphery of the cell; 3) the activation of an enzyme called Focal Adhesion Kinase; and 4) the deactivation of protein called mDia1 that organizes the cell skeleton. This research program will advance these findings, and provide insights that will resolve basic questions regarding the molecular signaling processes that focus force and shape strain in the cell during extracellular matrix assembly. The principal investigator has produced novel tools, assembled a world-class multidisciplinary team, and employed outstanding resources for cellular and molecular imaging and force measurements that will enhance the scientific impact of the work. Broader Impacts This project will further efforts to build a challenging and supportive interdisciplinary environment in which students of diverse backgrounds can build critical thinking and presentation skills, and develop new tools that benefit the scientific community. Fruits of these efforts to date include the following: the promotion and inclusion of a diverse group of students and postdoctoral trainees including female and underrepresented minority groups in hypothesis-driven and discovery science; the initiation of an interactive joint lab meeting incorporating labs from Biochemistry, Biophysics, Cell Biology, and Engineering at Johns Hopkins in order to provide opportunities for building presentation and experimental design skills for students in a collaborative forum with multiple scientific perspectives; research collaborations including an NSF Instrumentation grant that has recently made a new, state-of-the-art atomic force microscope available to the wider Hopkins community; innovation in research ethics teaching; and the provision of new image processing tools for free download for unrestricted use by the scientific community that are now in use for investigators across the U.S. and in France, India, and Australia. The current research project will advance these endeavors. An opportunity to learn new techniques for the measurement of interactions between signaling molecules will be created through a new collaboration with a research team at the Van Andel Research Institute. Further interdisciplinary and inter-institutional collaborations will be bolstered by this project through interactions with a matrix and cell biology lab at Duke University, and with biophysics and biology labs at Johns Hopkins. The breadth of expertise and resources that are available in this group will optimize the success of the research program, and ensure wide exposure of students to multiple disciplines. The breadth and synergy of the program will forge new links between divergent disciplines and provide other investigators with novel paradigms at the interface between physics and biology.