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

项目摘要/摘要 拟议的多样性补充是为了支持职业生涯早期的教员詹妮特·尚博士，她是一名中国人 斯克里普斯学院W.M.Keck科学系终身教职的第二年女性， 与圣地亚哥大学类似的以本科生为主的机构；反过来，也增强了多样性 与健康相关的劳动力。为期2年的补充计划的目的是推进和增强我们的R15资助 研究，并用技能、经验、独立性和强大的同事网络武装尚博士 研究人员在她的家乡机构建立了一个强大和创新的生物物理学研究计划。 具体地说，尚博士将为我们的母公司R15赠款的以下目标做出贡献和推进：(目标1)设计 在体外，活性细胞骨架网络表现出可调的活性，用于探测非平衡动力学和 流变性；(目标2)测定线状和环状DNA的运输和构象动力学 在活跃的细胞骨架网络内；(目标3)连接线性和环形DNA的时变空间分布 细胞骨架网络的性质和活性。本补充内容还将建立在目标3的基础上，超出最初的范围 由尚博士建造了一种结合了光镊子和光片的新型仪器 显微镜(LSM)用于同时测量细胞骨架网络力学和 大分子(即DNA)动力学。这一仪器和方法的进步将反过来取代尚博士的 处于生物医学和生物物理研究前沿的研究实验室，吸引新的外部合作者，并设立 作为一名独立研究人员，她走上了一条强劲的轨道。 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 和活性细胞骨架网络中线形和环状DNA的构象动力学；(目标S2)建立 新型荧光光片显微镜(LSM)和测力光钳(OT)相结合的DR 尚的实验室为我们的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 在张博士的家庭机构进行研究计划；(目标S3)进行有助于目标1-3的实验 父母R15和仪器在AIM S2的Sheung博士的实验室建立。 我们将在与尚博士建立的牢固的指导和合作关系的基础上， 帮助她实现短期和长期的职业目标。短期目标包括：(一)获得专门知识 在她自己的实验室里建造一个光片显微镜和光镊子；(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：(1)建立富有成效的独立跨学科研究 可供多学科本科生使用的课程，(Ii)建立外部合作者网络， 以及(Iii)使她所在部门的高级生物物理实验室课程现代化。

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.