| 研究概要 |
Cells and stress fibers (SFs) within the cells change their orientation in response to cyclic stretch. It has been reported that cyclic stretch-induced reorientation of SFs occurred as a result of the disassembly of pre-existing SFs. However, an elusive problem is the molecular mechanism underlying the disassembly of SFs. In 2013, to figure out the mechanism, first, the disassembly of SFs in cells exposed to cyclic stretch with different shortening rates was investigated. Disassembly of SFs in cells exposed to cyclic stretch with fast shortening phases increased significantly compared to cells under static conditions and exposed to cyclic stretch with slow shortening phases. In addition, the threshold rate of shortening for SF disassembly is comparable to the movement speed of nonmuscle myosin II (NMII), which is a motor protein moving along actin filaments. Second, mutants were transfected into cells to suppress cofilin or NMII activity and the change of SFs in cells exposed to cyclic stretch was observed. Although it has been reported that cofilin involved in SFs in shortened cells, we found that SF disassembly also occurred in cells exposed to cyclic stretch even in cofilin-inactivated cells. On the other hand, SFs in NMII-inactivated cells did not disassemble prominently even with cyclic stretch with a fast shortening rate. These results suggest that NMII should be the primary molecular mechanism involved in the SF disassembly in cells exposed to cyclic stretch.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
There are the equipments necessary to carry out the research efficiently and effectively, including stretching apparatus with programmable controller, confocal laser scanning microscope, inverted microscope with ORCA-2 CCD camera, live cell observation system, incubators, and so on. In addition, apparatuses for micropatterning are available anytime. I attended academic conferences and presented the research achievements and had discussion with the researchers of the previous studies on SF disassembly. The research was done with two research collaborators: Associate Professor Shinji Deguchi (Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology), and Ph. D. Tsubasa S. Matsui (Department of Biomolecular Sciences, Tohoku University and, Research Fellow of the Japan Society for the Promotion of Science). They have research expertise in studying the mechanical properties of SFs, and provided me some inhibitors or plasmids to suppress or enhance the activity of specific proteins, such as the cofilin and myosin activity. In addition, they played an active role in complementing the skills on experiments such as the live cell imaging technique, and gave me potentially very academically valuable advice, which thereby exponentially increased the research outputs.
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| 今後の研究の推進方策 |
The results of the study in 2013 suggest that SF disassembly was dependent on the rate of shortening. In addition, the hypothesis that nonmuscle myosin II was the molecular mechanism responsible for the SF disassembly exposed to cyclic stretch was confirmed. However, the real-time images of the process of SF disassembly in cells during cyclic stretch have not been observed. Seeing is believing. Therefore, the mission in 2014 is to specify the pattern of SF disassembly in cells during the entire process of cyclic stretch: pattern I: unbundling and disassembling into intact sarcomeric units of SFs induced by NMII detachment or, pattern II: directly depolymerizing into G-actin monomers induced by an actin-depolymerizing factor (ADF)/cofilin?
In previous studies using stretch chambers purchased from STREX (Osaka, Japan), time-lapse images of SFs in cells exposed to cyclic stretch cannot be taken automatically during the entire process of stretching for the following reasons: 1) the vertical position of the chamber membrane was shifted during the stretch, and 2) when the cells were exposed to 10% (or above) uniaxial cyclic stretch, the targeting cells were moved out of the microscope field and therefore, the real-time imaging of SFs in live cells at high resolution cannot be obtained. In 2014, I will fabricate a device, with which I can image the live cells transfected with EYFP-actin in real-time mode without stopping the stretch apparatus. With these images, the pattern of SF disassembly will be observed simply.
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