Cells universally rely on receptor proteins, present on the cell surface, to sense the environment and respond appropriately. Cells tune the strength and specificity of responses by changing the number of receptors available on the surface, by transporting them to and from compartments inside the cell. Therefore, how receptor transport is regulated is a fundamental question that is critical to understanding how cells respond and adapt to their environment. This project addresses this question by integrating direct visualization of the dynamics of receptor movement in living cells in real time, quantitative modeling, and molecular genetics, to test a mechanistic model for how receptor transport is regulated and how this regulation influences cellular responses. Through its broader impacts, this project offers an opportunity to train undergraduate and graduate students in using microscopy to address mechanistic questions, and to iteratively integrate microscopy, quantitative data analysis, and modeling to address cell biological questions. Further, to reach a broader community and promote the use of quantitative imaging in education at an early stage, the PI will actively introduce the tools developed and the information obtained to both science-focused and general public forums, including among students and teachers in high schools with underrepresented minorities, and in lifelong learning centers, in the Pittsburgh area.
The endosome serves as the primary sorting station for proteins, including signaling receptors, that are internalized from the cell surface. Of the four main pathways taken by internalized receptors in the endosome, three - bulk recycling, degradation, and trafficking to the Golgi - have been extensively studied, and the mechanisms identified have each defined major new directions in cell biology. This project addresses the fourth and comparatively less studied pathway - sequence-dependent recycling. Many signaling receptors are restricted to this pathway, but how and why are not known. A major hurdle in studying this process has been the lack of direct assays to visualize and analyze receptor sorting in real time. Emerging data indicate that endosomal sorting relies on kinetic principles, further emphasizing this. Therefore, the research goals of this project are to develop and use quantitative live-cell imaging approaches to directly visualize the sorting of B2AR, a member of the largest family of signaling receptors, as a prototype for sequence-dependent recycling, and to integrate the data obtained into quantitative models to test a mechanistic hypothesis for receptor sorting. By using approaches from whole-cell to single-event imaging, the project will test the hypotheses that signaling receptors are actively restricted to sequence-dependent recycling by their interactions with the putative endosomal complex identified, and that these interactions serve as control points for signaling pathways to reprogram receptor trafficking and signaling. The educational goal of this project is to promote quantitative microscopy as a mechanistic tool and train graduate and undergraduate students in methods in quantitative cell biology.
