Browsing by Subject "AID2"

Tropomyosins (Tpm) are coiled-coil proteins, which wind around actin filaments to form head-to-tail oligomers. Tpms control actin filament growth, movement and interactions with other actin-binding proteins like myosins and cofilin. Tpms play essential role in the construction and stabilization of complex three-dimensional actomyosin contractile structures called stress fibers, because Tpms regulate structural and functional attributes of actin filament populations. In mammals, there are 4 TPM genes encoding above 40 TPM variants, giving rise to many functional Tpm protein isoforms. They are responsible for several physiological mechanisms in cell such as morphogenesis, cytokinesis, vesicle transportation, metabolism, motility, organ development, and signaling. Even though several studies have been conducted to determine structures and functions of various Tpms, many questions are still to be answered about Tpm2.1 and its significance in cells. So far, Tpm2.1 isoform has been a difficult protein to study due to poor success rate at achieving its complete depletion from the cell. Its involvement in cytokinesis, cell movement, cancer progression, and association with mechanosensing ability of cells were recently reported, and this raised the interest of researchers to focus on unveiling its precise cell biological function. Conditional deletion, degradation or inactivation of a protein helps to determine its function in cells. A new revised Auxin Inducible Degron version-2 (AID2) approach employs the cell´s own ubiquitin mediated protein degradation process, ensuring efficient and rapid depletion of target protein by the help of expressing OsTIR1(F74G) auxin-receptor mutant in presence of 5-phenyl-indole-3 acetic acid (5-Ph-IAA) ligand. In this study, we established a pipeline to identify Tpm2.1’s functions using AID2 technology by integrating OsTIR1(F74G) mutant at AAVS1 locus of the homozygous knock-in U2OS clones, containing mAID-msGFP2-TPM2.1 fusion insert at their endogenous TPM2.1 locus. We aimed to deplete Tpm2.1 from cells using this approach by inducing with 5-Ph-IAA and observe the direct, immediate phenotypes during Tpm2.1 degradation. We succeeded in achieving almost complete Tpm2.1 depletion. By this approach, we revealed that Tpm2.1 controls actin reorganization, stress fiber stability, and maturation of focal adhesions in cultured cells. To our knowledge, Tpm2.1 is the first actin-binding protein to be studied using AID2 approach, and the promising outcome brings hope to study other complicated actin-regulating proteins with this approach.