Investigating the dynamics and mechanics of CLASP-mediated microtubule-actin interactions by combining in vitro reconstitution with studies in primary lung fibroblasts

通过将体外重建与原代肺成纤维细胞研究相结合，研究 CLASP 介导的微管-肌动蛋白相互作用的动力学和机制

• 批准号: 10338099
• 负责人: Nicole Christina Rodgers
• 金额: $ 3.08万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10338099
• 关键词: Actins; Affect; Atomic Force Microscopy; Award; Binding; Biochemical; Biochemistry; Biomedical Engineering; Breathing; Cardiovascular Diseases; Cardiovascular system; Cell physiology; Cells; Cellular Morphology; Cellular biology; Cicatrix; Collaborations; Connective Tissue; Cytoskeleton; Developmental Biology; Ensure; Event; Extracellular Matrix; F-Actin; Faculty; Family; Fellowship; Fibroblasts; Fibrosis; Fluorescence Microscopy; Funding; Growth; Health; Human; Impairment; In Vitro; Individual; Investigation; Laboratories; Lead; Link; Lung; Lung diseases; Maintenance; Measures; Mechanics; Mediating; Microfilaments; Microfluidics; Microtubule-Associated Proteins; Microtubules; Molecular; Myocardial Infarction; Neurons; Organ; Physics; Plus End of the Microtubule; Polymers; Process; Production; Property; Protein Family; Proteins; Pulmonary Fibrosis; Reporting; Role; Science; Stress Fibers; Structure; Techniques; Testing; Tissue Expansion; Training; base; cell motility; coronary fibrosis; crosslink; experimental study; insight; interdisciplinary approach; live cell imaging; mechanical properties; member; overexpression; physical science; protein crosslink; reconstitution; wound healing

PROJECT SUMMARY/ABSTRACT The ability of fibroblasts to produce force is essential for maintenance of the extracellular matrix, cell motility, and wound repair. Overactivated fibroblasts are linked to cardiovascular and pulmonary fibrosis due to an excess of connective tissue causing scarring. This in turn results in an inability for proper tissue expansion and can lead to myocardial infarction and difficulty in breathing. Force production by fibroblasts, in part, is driven by interactions between microtubule and actin cytoskeletons, coordinated by crosslinking proteins to ensure proper cell migration. Members of the CLASP (Cytoplasmic Linker Associated Protein) family of proteins have been implicated in the cytoskeletal crosstalk in the context of fibroblast function. However, the specific dynamic and mechanical interactions between microtubules and actin mediated by CLASPs remain elusive. This study will use in vitro reconstitution of microtubules and actin with purified proteins and investigations in human lung fibroblasts to elucidate interactions between microtubules and actin mediated by CLASP2. Preliminary results demonstrate that CLASP2⍺ directly interacts with actin in vitro, with a stronger colocalization with bundled actin filaments, and facilitates interactions between microtubules and actin. First, the preferential actin substrate for CLASP2-mediated crosslinking of actin with microtubules will be characterized. Then, the individual and global CLASP2-mediated interactions with dynamic microtubules and actin will be quantified in vitro and in human lung fibroblasts. Second, the mechanical properties of CLASP2-crosslinked microtubule-actin polymers will be characterized by using microfluidic flow to investigate the strength and mechanical stability in vitro. Furthermore, atomic force microscopy will be used to measure the elastic properties of human lung fibroblast cells. The combination of in vitro reconstitution and experiments in human lung fibroblasts will elucidate the biochemical and mechanical mechanisms underlying microtubule-actin coordination by CLASP2. This fellowship award will not only fund the proposed project to elucidate the dynamics and mechanics of microtubule and actin interactions in the context of human fibroblasts but will also support the applicant’s training in interdisciplinary science. The project involves a collaboration between two laboratories with complementary expertise in biochemical reconstitution and cell biology. The applicant will apply her background in the physical sciences, along with dedicated support from the sponsor, co-sponsor, and thesis committee, composed of faculty in Cell and Developmental Biology, Biochemistry, Physics, and Biomedical Engineering, to complete the proposed project. Elucidating the mechanisms underlying fibroblast force production by using multidisciplinary approaches will provide an important understanding of processes that drive cardio and pulmonary fibrosis.

项目摘要/摘要 成纤维细胞产生力的能力对于维持细胞外基质，细胞至关重要 运动性和伤口修复。过度活化的成纤维细胞与由于心血管和肺纤维化有关 过量的连接组织会导致疤痕。反过来，这导致无法进行适当的组织扩张 并可能导致心肌梗塞，呼吸困难。成纤维细胞产生的力部分是驱动的 通过微管与肌动蛋白细胞骨架之间的相互作用，通过交联蛋白协调以确保 正确的细胞迁移。蛋白质蛋白质家族的扣（细胞质接头蛋白）的成员具有 在成纤维细胞功能的背景下与细胞骨架串扰有关。但是，特定的动态 微管与链球菌介导的肌动蛋白之间的机械相互作用仍然难以捉摸。这项研究 将使用纯化蛋白质的微管和肌动蛋白的体外重构，并在人肺中进行研究 成纤维细胞可阐明微管之间的相互作用和clasp2介导的肌动蛋白。初步结果 证明clasp2⍺在体外与肌动蛋白直接相互作用，与捆绑肌动蛋白更强的共定位 细丝，并促进微管与肌动蛋白之间的相互作用。首先，首选的肌动蛋白底物 将表征肌动蛋白与微管的CLASP2介导的交联。然后，个人和全球 CLASP2介导的与动态微管和肌动蛋白的相互作用将在体外和人肺中进行定量 成纤维细胞。其次，CLASP2-跨链接微局部 - 肌动蛋白聚合物的机械性能是 通过使用微流动流来研究体外强度和机械稳定性的特征。此外， 原子力显微镜将用于测量人肺成纤维细胞的弹性特性。这 体外重构和实验在人肺成纤维细胞中的结合将阐明生化 和通过clasp2进行微管 - 肌动蛋白协调的机械机制。这个奖学金奖将 不仅为拟议项目提供了阐明微管和肌动蛋白相互作用的动力学和力学 在人类成纤维细胞的背景下，也将支持申请人在跨学科科学方面的培训。这 项目涉及两个实验室之间具有生化专业知识的实验室的合作 重构和细胞生物学。申请人将把她的背景应用于物理科学，以及 赞助商，共同发起人和论文委员会的专门支持，由细胞中的教职员工组成 发育生物学，生物化学，物理和生物医学工程，以完成拟议项目。 使用多学科方法阐明成纤维细胞力产生的机制将 对驱动心脏和肺纤维化的过程提供重要的理解。