Molecular Regulation of Matrix Assembly Mechanics

Johns Hopkins University, Baltimore MD

Investigators

Abstract

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.