The major goal of this research is to contribute to our mechanistic understanding of how cells communicate with each other and the outside environment. Communication among cells is critical for most, if not all, living systems and regulates processes including cell migration, cell division, growth, and death. One way this occurs is though a process called endocytosis whereby molecules are transported into the cell. Endocytosis requires an intricate coordination of proteins inside the cell as well as physical changes to the surface of the cell. The deformation of the cell's surface is central to endocytosis, yet there are few tools that can dissect these physical changes that are necessary for endocytosis. One reason this is difficult to study is that this processes is dynamic and occurs on the nanoscale, beyond the resolution of traditional microscopy. This project will advance the study of nanometer scale cell surface dynamics by developing and applying new methods to measure and manipulate the process of endocytosis in living cells. Many biological processes occur on this scale, and the methods developed in this research will be widely applicable across a range of biological systems including virus entry and budding, cell migration, and neurobiology. In addition to contributing to understanding cellular processes, the effort will include training students from K-12 through graduate school on microscopy and the value of multi-disciplinary approaches in understanding cellular processes. The goal of this research is to understand the dynamics of endocytic vesicle assembly and the interconnected roles played by the proteins involved, as well as the physical changes to plasma membrane shape. A recently developed fluorescence microscopy technique is uniquely suited to imaging nanometer dynamics of vesicle formation in real-time in living cells. In Aim 1 the novel microscopy technique is used to measure endocytic pit formation dynamics upon stimulation with EGF. This experimental platform will then be used to dissect the protein dynamics during pit formation, mapping the correlation between protein dynamics and plasma membrane morphology (Aim 2). Finally, a strategy will be developed to physically stall endocytosis utilizing a tethered ligand. This platform will be used to explore curvature sensing and curvature inducing proteins (Aim 3). This research will yield a quantitative description of ligand internalization dynamics and the interplay between protein recruitment and membrane morphology on a millisecond time scale. Completion of this research will shed light on previously undiscovered mechanisms of plasma membrane dynamics and signaling that may revolutionize the way cellular communication is understood. Further, it will provide new tools to broadly improve the understanding of plasma membrane dynamics in cell signaling and homeostasis. In addition to providing K-12 though graduate students with a rich educational experience, the PI will make available to the community any software that is produced and which is useful to others in achieving high resolution data. This award is supported jointly by the Division of Molecular and Cellular Biosciences and the Physics of Living Systems program in the Division of Physics
