Cells are the basic units of life. They function by receiving signals from the outside and processing these signals to regulate cellular behavior. Signal processing is a complex process involving tens if not hundreds of cellular components (referred to as signaling intermediates) that must interact with each other to regulate cellular behavior. The amounts of such signaling intermediates inside each cell vary as a function of time after receiving a signal from the outside and as a function of their location in the cell. Jointly, these are referred to as spatiotemporal patterns. Spatiotemporal patterns are critical for understanding signaling efficiency in live cells, as enrichment of two proteins in the same location at the same time increases the likelihood that they will interact, i.e. their efficiency. Thus, an understanding of spatiotemporal pattering is essential if we are to unravel the complex interaction network of tens to hundreds of signaling intermediates, a great challenge in current biology. The goals of this project are: 1) To acquire comparative image data sets on many protein signaling intermediates under varying conditions of cellular activation and to develop and apply image analysis methods to accurately define the amount and location of the signaling intermediates within a cell (the T lymphocyte). In addition, these methods will be applied to define how the amount and location of these signaling intermediates vary with time as T cells respond to the different stimuli. 2) Because the data and methods that will be developed in this project will be of general interest and value for understanding cellular signaling, they will be made publicly available on servers. Other investigators will be given the opportunity to use these methods to analyze their image data. T cell microscopy experiments will be executed for collaborators, and the visual appeal of imaging data will be used to enrich high school education.
Broader impacts: In addition to providing the research community access to the resources and data that will be developed, this project will engage high school, graduate and medical students, as well as two postdoctoral fellows. Undergraduate students from around the US will be given opportunities to work on this project through the University of Texas Southwestern Summer Undergraduate Research Fellowship program (which places an emphasis on recruiting underrepresented minorities). Dr. Murphy will train and mentor minority students through a Research Experiences for Undergraduates program at Carnegie-Mellon University.
The combination of fluorescent protein tagging and fluorescence microscopy permits movies to be collected of the intracellular distributions of different proteins in live cells as they undergo various cellular processes. However, computational methods are required in order to analyze these movies and produce detailed models of how different proteins. This grant supported development and testing of such methods and their use to analyze movies collected through a parallel grant. The process studied was T-cell synapse formation, which is critical to functioning of the immune system. The results results revealed which proteins show altered distributions when T cell signaling was partially blocked, indicating that those proteins are important regulators of T cell function. The models developed will be useful for building cell simulations of the process of T cell signaling, and the methods will also be useful for future studies of changes in protein location in other cell processes. The methods have been incorporated into open source software and made available to the research community. The grant also supported training and mentoring of a number of Ph.D. students. The grantee gave many lectures and organized a tutorials and a workshops to educate other scientists about these new methods and the results obtained.