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

项目摘要/摘要：获取分子时空定量信息的方法 细胞中的机制是理解为什么细胞行为在疾病进展过程中发生改变的基础 疾病。这项提议的总体目标是开发一个灵活的成像平台，用于同时进行多个 彩色三维单分子超分辨成像和三维单粒子多染色体跟踪 整个活的哺乳动物细胞中具有高时空分辨率的基因座，并使用这个平台来 确定核层蛋白、前层蛋白A突变的分子机制和相互作用 蛋白质导致Hutchinson-Gilford Progeria综合征(HGPS)。患有HGPS的儿童年龄迅速，死亡率中值为 13岁，没有治疗选择。我们的假设是抑制翻译后修饰的药物 突变的Prelamin A通过改变其空间分布和分子相互作用来降低其毒性。 具体目标是：1)开发一种在整个直播过程中以3D方式跟踪多个染色体位置的策略 哺乳动物的细胞核。结合光片照明、用于3D检测的PSF工程和纳米体 阵列标签将导致在从毫秒到小时的时间范围内进行数十纳米精度的跟踪。 2)扩展了多色3D SR成像的成像平台，并确定了 抑制前层蛋白A的法尼化和甲基化。成像平台将增加多色功能 和分析算法，以及前层蛋白A分布的变化和药物治疗与非药物治疗的相互作用 将会被测量。3)确定改变的(前)层蛋白A分布如何影响染色质动力学，以及 HGPS和对照细胞之间的相互作用。蛋白质分布和核动力学之间的关联将是 通过结合目标1和目标2中的策略并扩展活细胞3D SR成像平台来映射。 该项目的成功完成将提供一个创新和强大的成像平台，将 对未来的生物医学研究产生重大影响。这一结果将对儿童具有重要意义 通过对HGPS的分子机制的推断，提出了一种新的潜在的治疗策略。 最终，这个项目也将有助于我成为一名独立研究员的长期职业目标 在学术界，推动细胞成像的前沿，以解决生物物理、生物化学和医学方面的问题 受当前技术的限制。我的职业发展计划包括不同的培训主题，重点是 提高我的生物技术技能，这将使我未来的独立研究。培训将会 在优秀的导师和顾问的指导下进行，这些导师和顾问已被选为支持和 在拟议研究的所有步骤中提供指导。斯坦福大学提供一整套教育资源 包括正式的课程和研讨会，以及出色的研究环境，以供进一步培训，如 这是必要的。因此，NIH K99/R00奖项将使我获得最先进的培训、专业知识、 和世界级的技能发展成为一名成功的独立调查员。

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.