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

早衰症疾病及治疗机制的三维超分辨率成像与追踪

• 批准号: 10396130
• 负责人: Anna Karin Eva Gustavsson
• 金额: $ 24.9万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396130
• 关键词: 3-Dimensional; Academia; Address; Affect; Age; Algorithmic Analysis; Award; Binding; Biochemistry; Biological; Biomedical Research; Biophysics; Cell Nucleus; Cell physiology; Cells; Characteristics; Child; Chromatin; Collaborations; Color; Cysteine; DNA; Data; Detection; Development Plans; Disease; Engineering; Environment; Enzymes; Event; Foundations; Future; Goals; Health; Hour; Image; Individual; Kinetics; Knowledge; Label; Laboratories; Lamin Type A; Light; Lighting; Link; Mammalian Cell; Maps; Measurement; Measures; Medicine; Mentors; Methods; Methylation; Molecular; Motion; Mutate; Mutation; Nuclear; Nuclear Lamina; Performance; Pharmaceutical Preparations; Pharmacotherapy; Post-Translational Protein Processing; Progeria; Proteins; Proto-Oncogene Proteins c-akt; Research; Research Personnel; Resolution; Scheme; Spatial Distribution; Structure; Syndrome; Techniques; Therapeutic; Time; Toxic effect; Training; United States National Institutes of Health; Universities; Work; Yeasts; associated symptom; career; career development; cell behavior; cellular imaging; cost efficient; education resources; farnesylation; flexibility; fluorophore; frontier; imaging platform; innovation; millisecond; mutant; nanobodies; nanometer; nanometer resolution; nanoscale; normal aging; novel; particle; prelamin A; protein distribution; single molecule; skills; spatiotemporal; treatment strategy; 3-Dimensional; Academia; Address; Affect; Age; Algorithmic Analysis; Award; Binding; Biochemistry; Biological; Biomedical Research; Biophysics; Cell Nucleus; Cell physiology; Cells; Characteristics; Child; Chromatin; Collaborations; Color; Cysteine; DNA; Data; Detection; Development Plans; Disease; Engineering; Environment; Enzymes; Event; Foundations; Future; Goals; Health; Hour; Image; Individual; Kinetics; Knowledge; Label; Laboratories; Lamin Type A; Light; Lighting; Link; Mammalian Cell; Maps; Measurement; Measures; Medicine; Mentors; Methods; Methylation; Molecular; Motion; Mutate; Mutation; Nuclear; Nuclear Lamina; Performance; Pharmaceutical Preparations; Pharmacotherapy; Post-Translational Protein Processing; Progeria; Proteins; Proto-Oncogene Proteins c-akt; Research; Research Personnel; Resolution; Scheme; Spatial Distribution; Structure; Syndrome; Techniques; Therapeutic; Time; Toxic effect; Training; United States National Institutes of Health; Universities; Work; Yeasts; associated symptom; career; career development; cell behavior; cellular imaging; cost efficient; education resources; farnesylation; flexibility; fluorophore; frontier; imaging platform; innovation; millisecond; mutant; nanobodies; nanometer; nanometer resolution; nanoscale; normal aging; novel; particle; prelamin A; protein distribution; single molecule; skills; spatiotemporal; treatment strategy

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.

项目摘要/摘要：获得有关分子的定量时空信息的方法 细胞中的机制对于理解为什么细胞行为在进展中改变了为什么 疾病。该提案的总体目的是为同时多的多种成像平台开发灵活的成像平台 颜色3D单分子超分辨率（SR）成像和3D单粒子跟踪多个染色体 在整个活哺乳动物细胞中具有高时空分辨率的基因座，并使用此平台来 确定核椎板蛋白，预胺A的分子机制和相互作用。 蛋白质会导致Hutchinson-Gilford Progeria综合征（HGP）。 HGPS年龄迅速的孩子，死于中位数 13岁，没有治疗选择。我们的假设是抑制翻译后修饰的药物 突变的预胺A的A通过改变其空间分布和分子相互作用来降低其毒性。 具体目的是：1）制定一种策略，以跟踪整个Live的3D染色体基因座 哺乳动物细胞核。结合光片照明，用于3D检测的PSF工程和纳米机构 阵列标签将导致跟踪数十纳米的精度，从毫秒到几个小时。 2）扩展多色3D SR成像的成像平台，并确定分子后果 抑制前A的Farnesylation和A.甲基化。多色能力将添加到成像平台中 和分析算法，以及预胺A分布和与药物治疗的相互作用的变化 将测量。 3）确定染色质动力学如何受（前）层粘连蛋白A分布和 HGP和对照细胞中的相互作用。蛋白质分布与核动力学之间的相关性将是 通过将AIM 1和2中的策略结合在一起，并扩展了实时3D SR成像的平台。 该项目的成功完成将提供一个创新且功能强大的成像平台，将 未来对生物医学研究产生巨大影响。结果对儿童具有重要意义 通过推断综合征的分子机制和新的潜在治疗策略的分子机制，使用HGP。 最终，该项目还将促进我成为独立研究人员的长期职业目标 在学术界，推动细胞成像的前沿以解决生物物理，生物化学和医学的问题 受当前技术的限制。我的职业发展计划包括各种培训主题，重点是 提高我在生物技术方面的技能，这将使我未来的独立研究能够。训练会 在被选为支持的优秀导师和顾问的指导下进行 拟议研究的所有步骤中的指导。斯坦福大学提供了一套完整的教育资源 包括正式的课程和研讨会，以及一个出色的研究环境，以进一步培训 必要的。因此，NIH K99/R00奖将使我获得最先进的培训，专家知识， 和世界一流的技能，成为成功的独立研究者。