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.
Fibroblast motility is a cellular process that relies on coordinated interactions between the microtubule and actin cytoskeletons, and when impaired, can result in cardiac and pulmonary fibrosis. This interdisciplinary project combines in vitro reconstitution approaches using purified protein components with studies in human lung fibroblasts to elucidate the dynamics and mechanics of microtubule-actin interactions mediated by a crosslinking protein, Cytoplasmic Linker Associated Protein (CLASP). The results from this study will provide insight into the molecular mechanisms underlying fibroblast function, ultimately contributing to our broader understanding of processes leading to cardiovascular and pulmonary disease.
