Three-dimensional super-resolution imaging and tracking of disease and treatment mechanisms of progeria

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Project Summary/Abstract: Methods to obtain quantitative spatiotemporal information about molecular mechanisms in cells are fundamental for the understanding of why cellular behavior is altered in the progress of disease. The overall objective of this proposal is to develop a flexible imaging platform for simultaneous multi- color 3D single-molecule super-resolution (SR) imaging and 3D single-particle tracking of multiple chromosomal loci with high spatiotemporal resolution throughout entire live mammalian cells, and to use this platform to determine molecular mechanisms and interactions of the nuclear lamina protein, prelamin A. Mutations in this protein causes Hutchinson-Gilford Progeria Syndrome (HGPS). Children with HGPS age rapidly, die at a median age of 13, and have no therapeutic options. Our hypothesis is that drugs inhibiting posttranslational modifications of mutated prelamin A reduces its toxicity by altering its spatial distribution and molecular interactions. The specific aims are to: 1) Develop a strategy to track multiple chromosomal loci in 3D throughout live mammalian cell nuclei. Combining light sheet illumination, PSF engineering for 3D detection, and nanobody array labels will result in tracking with tens of nanometers precision over time scales from milliseconds to hours. 2) Extend the imaging platform for multi-color 3D SR imaging and determine the molecular consequences of inhibiting farnesylation and methylation of prelamin A. Multi-color capability will be added to the imaging platform and analysis algorithm, and changes in prelamin A distribution and interactions with and without drug treatments will be measured. 3) Determine how chromatin dynamics are affected by altered (pre)lamin A distribution and interactions in HGPS and control cells. Correlation between protein distributions and nuclear dynamics will be mapped by combining the strategies in Aim 1 and 2 and extending the platform for live-cell 3D SR imaging. The successful completion of this project will provide an innovative and powerful imaging platform that will have dramatic impact on biomedical research in the future. The results will be of great significance for children with HGPS by deducing molecular mechanisms of the syndrome and of a new potential treatment strategy. Ultimately, this project will also facilitate my long-term career goal of becoming an independent researcher in academia, pushing the frontiers of cell imaging to address questions in biophysics, biochemistry, and medicine limited by current techniques. My career development plan includes diverse training topics with a focus on increasing my skills in biological techniques, which will enable my future independent research. The training will take place under the guidance of excellent mentors and advisors who have been selected for support and guidance in all steps of the proposed research. Stanford University offers a complete set of educational resources including formal coursework and seminars, and an outstanding research environment for further training as necessary. Thus, the NIH K99/R00 award would allow me to gain state-of-the-art training, expert knowledge, and world class skills to develop into a successful independent investigator.