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.

项目概要/摘要 成纤维细胞产生力的能力对于维持细胞外基质、细胞 过度活化的成纤维细胞与心血管和肺纤维化有关。 过多的结缔组织会导致疤痕，从而导致组织无法适当扩张。 并可导致心肌梗塞和呼吸困难，部分是由成纤维细胞产生的力驱动的。 通过微管和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 CLASP（细胞质连接蛋白）家族的成员具有正确的细胞迁移能力。 与成纤维细胞功能背景下的细胞骨架串扰有关，但是具体的动态。 CLASP 介导的微管和肌动蛋白之间的机械相互作用仍然难以捉摸。 将利用纯化蛋白体外重建微管和肌动蛋白，并在人肺中进行研究 成纤维细胞阐明 CLASP2 介导的微管和肌动蛋白之间的相互作用。 证明 CLASP2⍺ 在体外直接与肌动蛋白相互作用，与捆绑的肌动蛋白具有更强的共定位性 首先，肌动蛋白的优先底物。 然后，对 CLASP2 介导的肌动蛋白与微管的交联进行个体和整体的表征。 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量化 其次，CLASP2 交联的微管-肌动蛋白聚合物的机械性能。 通过使用微流体流动来研究体外强度和机械稳定性。 原子力显微镜将用于测量人肺成纤维细胞的弹性特性。 体外重建和人肺成纤维细胞实验的结合将阐明生化 CLASP2 微管-肌动蛋白协调的机械机制。 不仅资助拟议的项目来阐明微管和肌动蛋白相互作用的动力学和机制 在人类成纤维细胞的背景下，还将支持申请人的跨学科科学培训。 该项目涉及两个具有生物化学专业知识互补的实验室之间的合作 申请人将应用她在物理科学方面的背景以及 来自赞助商、共同赞助商和论文委员会（由 Cell 和 发育生物学、生物化学、物理学和生物医学工程，以完成拟议的项目。 使用多学科方法阐明成纤维细胞力产生的机制将 提供对驱动心脏和肺纤维化过程的重要理解。