Biomimetic cytoskeleton and advanced microscopy to reveal intracellular DNA dynamics and distributions

项目概要/摘要 建议的多元化补充是为了支持早期职业教师，珍妮特博士，一个中国人 妇女在她的第二年的终身教职职位在WM。斯克里普斯学院凯克科学系， 一个以本科为主的机构，类似于圣地亚哥大学;反过来，以提高多样性 与健康相关的劳动力。2年补充的目的是推进和加强我们的R15资助 研究，并以技能，经验，独立性和强大的研究员网络武装尚博士 研究人员在她的家乡机构建立一个强大的和创新的生物物理学研究计划。 具体而言，尚博士将致力于推动我们的R15补助金的以下目标：（目标1）设计 体外活性细胞骨架网络，其表现出探测非平衡动力学的可调活性， 流变学性质;（目的2）确定线性和环状DNA的运输和构象动力学 在活跃的细胞骨架网络中;（目的3）链接线性和环状DNA的时变空间分布 细胞骨架网络的性质和活动。本补编还将以目标3为基础，超出最初的范围 由Sheung博士建造一种新颖的仪器，结合了光学镊子（OT）和光片 显微镜（LSM）进行细胞骨架网络力学的同时测量， 大分子（即，DNA）动力学。这种仪器和方法的进步反过来又将使Sheung博士的 研究实验室在生物医学和生物物理研究的前沿，吸引新的外部合作者，并设置 她作为一名独立研究员走上了一条强大的道路。 为了实现这些目标，我们将追求三个目标：（目标S1）执行目标2实验，以确定运输 和线性和环状DNA在活性细胞骨架网络中的构象动力学;（目的S2）建立一个 新型组合荧光光片显微镜（LSM）和测力光镊（OT）在博士。 Sheung的实验室为我们的R15目标1-3提供独特的贡献，并建立一个强大的本科生驱动的 在常博士的家乡机构进行研究计划;（目标S3）进行有助于目标1-3的实验， 在Aim S2的尚博士实验室内安装了R15的原型和仪器。 我们将与尚博士建立稳固的指导和合作关系， 帮助她实现短期和长期的职业目标。短期目标包括：（一）获得专业知识 在她自己的实验室里建造光片显微镜和光镊;（ii）制定有效的战略， 在暑期研究项目中指导本科生和高中生;以及（iii）接受联邦资助 为了她的研究长期目标包括：（一）建立一个富有成效的独立的跨学科研究 程序访问本科生从多个学科，（ii）建立外部合作者的网络， 以及（iii）使她的家庭部门的高级生物物理学实验室课程现代化。

Project Summary/Abstract The proposed diversity supplement is to support early career faculty member, Dr. Janet Sheung, a Chinese woman in her 2nd year of a tenure-track faculty position in the W.M. Keck Science Department at Scripps College, a predominantly undergraduate institution similar to University of San Diego; and, in turn, to enhance the diversity of the health-related workforce. The aims of the 2-year supplement are to advance and enhance our R15-funded research, and to arm Dr. Sheung with skills, experience, independence, and a strong network of fellow researchers to establish a robust and innovative biophysics research program at her home institution. Specifically, Dr. Sheung will contribute to and advance the following Aims of our parent R15 grant: (Aim 1) Design in vitro active cytoskeleton networks that exhibit tunable activity for probing non-equilibrium dynamics and rheological properties; (Aim 2) Determine transport and conformational dynamics of linear and circular DNA within active cytoskeleton networks; (Aim 3) Link the time-varying spatial distributions of linear and circular DNA to cytoskeleton network properties and activity. This supplement will also build on Aim 3, beyond the initial scope of the grant, by Dr. Sheung building a novel instrument that combines optical tweezers (OT) and light-sheet microscopy (LSM) to perform simultaneous measurements of cytoskeleton network mechanics and macromolecule (i.e., DNA) dynamics. This instrumentation and method advance will, in turn, place Dr. Sheung’s research lab at the forefront of biomedical and biophysical research, draw in new external collaborators, and set her on a strong trajectory as an independent researcher. To achieve these goals, we will pursue three Aims: (Aim S1) Perform Aim 2 experiments to determine transport and conformational dynamics of linear and circular DNA within active cytoskeleton networks; (Aim S2) Build a novel combined fluorescence light-sheet microscope (LSM) and force-measuring optical tweezers (OT) in Dr. Sheung’s lab to provide unique contributions to our R15 Aims 1-3 and establish a robust undergraduate-driven research program at Dr. Sheung’s home institution; (Aim S3) Perform experiments that contribute to Aims 1-3 of the parent R15 with instrumentation established in Dr. Sheung’s lab in Aim S2. We will build on the strong mentoring and collaboration relationship we have established with Dr. Sheung, to help her achieve her short-term and long-term career goals. Short-term goals include: (i) gaining the expertise to construct a light-sheet microscope and optical tweezers in her own lab; (ii) developing strategies to effectively mentor undergraduate and high school students in summer research projects; and (iii) receiving federal funding for her research. Long-term goals include: (i) establishing a fruitful independent interdisciplinary research program accessible to undergraduates from multiple disciplines, (ii) building a network of external collaborators, and (iii) modernizing the advanced biophysics lab course in her home department.