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 成纤维细胞产生力量的能力对于维持细胞外基质细胞是必不可少的。 活动能力和伤口修复能力。成纤维细胞过度激活与心血管和肺纤维化有关，原因是 结缔组织过多导致结疤。这反过来又导致无法进行适当的组织扩张 并可能导致心肌梗死和呼吸困难。成纤维细胞产生的力量在一定程度上是由 通过微管和肌动蛋白细胞骨架之间的相互作用，通过交联蛋白的协调来确保 适当的细胞迁移。CLAP(细胞质连接蛋白相关蛋白)家族的成员有 在成纤维细胞功能的背景下与细胞骨架串扰有关。然而，具体的动态 微管和肌动蛋白之间由卡环介导的机械相互作用仍然难以捉摸。本研究 将利用纯化的蛋白质体外重建微管和肌动蛋白，并在人肺中进行研究 以阐明CLASP2介导的微管和肌动蛋白之间的相互作用。初步结果 证明CLASP2⍺在体外与肌动蛋白直接相互作用，与捆绑肌动蛋白有较强的共定位 细丝，促进微管和肌动蛋白之间的相互作用。首先，优先选择肌动蛋白底物 本课程将描述CLASP2介导的肌动蛋白与微管的交联性。然后，个人和全局 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被量化 成纤维细胞。第二，CLASP2-交联型微管-肌动蛋白聚合物的机械性能将是 采用微流体流技术对材料的体外强度和机械稳定性进行研究。此外， 原子力显微镜将被用来测量人肺成纤维细胞的弹性性质。这个 体外重建和人肺成纤维细胞实验相结合将阐明生物化学 以及CLASP2协调微管-肌动蛋白的机械机制。该奖学金将颁发给 不仅资助了拟议的项目，以阐明微管和肌动蛋白相互作用的动力学和机制 在人类成纤维细胞方面，还将支持申请者在跨学科科学方面的培训。这个 该项目涉及两个在生化方面具有互补专业知识的实验室之间的合作 重建和细胞生物学。申请者将申请她的物理科学背景，以及 来自发起人、共同发起人和论文委员会的全力支持，该委员会由Cell和 发展生物学、生物化学、物理学和生物医学工程，以完成拟议的项目。 用多学科方法阐明成纤维细胞力量产生的机制将 对推动心脏和肺纤维化的过程有重要的了解。