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

项目总结/摘要 成纤维细胞产生力的能力对于维持细胞外基质、细胞外基质和细胞外基质是至关重要的。 运动和伤口修复过度活化的成纤维细胞与心血管和肺纤维化有关， 导致疤痕的过多的结缔组织。这反过来又导致无法进行适当的组织扩张 并可导致心肌梗塞和呼吸困难。成纤维细胞产生力的部分原因是 通过微管和肌动蛋白细胞骨架之间的相互作用，通过交联蛋白质协调， 正常的细胞迁移CLASP（细胞质接头相关蛋白）蛋白家族的成员具有 在成纤维细胞功能的背景下，与细胞骨架串扰有关。然而，具体的动态 由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。本研究 将使用纯化的蛋白质在体外重建微管和肌动蛋白，并在人肺中进行研究 成纤维细胞阐明微管和肌动蛋白介导的CLASP 2之间的相互作用。初步结果 证明CLASP 2 β在体外直接与肌动蛋白相互作用，与成束的肌动蛋白具有更强的共定位 微丝，并促进微管和肌动蛋白之间的相互作用。首先，优先的肌动蛋白底物 CLASP 2介导的肌动蛋白与微管的交联将被表征。然后，个人和全球 CLASP 2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定量 成纤维细胞第二，CLASP 2交联的微管-肌动蛋白聚合物的机械性能将是 通过使用微流体流动来研究体外强度和机械稳定性。此外，委员会认为， 原子力显微镜将用于测量人肺成纤维细胞的弹性特性。的 体外重建和人肺成纤维细胞实验的结合将阐明生物化学 和机械机制的微管肌动蛋白协调CLASP 2。该奖学金将 不仅资助拟议的项目，以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，但也将支持申请人在跨学科科学的培训。的 该项目涉及两个实验室之间的合作，在生物化学方面具有互补的专业知识 重组和细胞生物学。申请人将应用她在物理科学的背景，沿着 来自赞助商，共同赞助商和论文委员会的专门支持，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。 通过使用多学科方法阐明成纤维细胞力产生的机制将 提供了驱动心脏和肺纤维化的过程的重要理解。

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.