| 研究実績の概要 |
Below is the highlight of progress we have made in FY 2019:
1. One of our primary goals is to visualize and 3D reconstruct the apical dome of epithelial cells during dorsal fold initiation in the gastrulating Drosophila embryo. The key components include the membranes, the microtubule cytoskeleton and its associated proteins, and the hypothetical counterbalancing mechanical structures at the apical membrane. Of these key components, we now have much improved membrane marker (3XmScarlet-CAAX), microtubule marker (EMTB-3XGFP), and an excellent EB1 marker to mark the microtubule plus end (ncd-EB1-GFP).
2. In our published work (Takeda at al 2018 NCB) we hypothesized that the microtubule pushing forces are counterbalanced at the apical membrane. We have tested one of candidates that we previously proposed, the Spectrin membrane skeleton, and found that reduction of the main component the Spectrin system, alpha-Spectrin, by RNAi knockdown results in delayed apical dome decent. These preliminary data are supportive of the idea that the Spectrin membrane skeleton system is involved in the mechanics of the apical counterbalancing force.
3. Through our collaboration with Drs. Mitsusuke Tarama and Tatsuo Shibata, tremendous progress has been made in implementinh a disordered filamentous network model coupled with spatial confinement simulating the apical membrane. The network model had provided preliminary support that minus-end filament anchorage coupled with the minus-end directed motor can produce a collective pushing force acting on the apical membrane.
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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
As summarized above, we have made crucial progress in all three key directions of the research. First, with a newly constructed set of imaging markers, we will be in a better position to visualize, quantitate and 3D reconstruct the molecular dynamics and structure that define and control and apical dome mechanics. Second, the genetic data that support the idea that the Spectrin system is involved in the apical counterbalancing force is a major breakthrough as this is the missing component that have previously been predicted. Lastly, the theoretical modeling aspect of this research has also seen its breakthrough in the past year. The mathematical model that Dr. Tarama has implemented will have a major impact both within the specific context of the apical dome research as well as on the theoretical understanding of membrane/cytoskeleton interplay in general.
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| 今後の研究の推進方策 |
Going forward in FY2020, we expect the research will pick up speed in all three areas mentioned above.
1. On the imaging front, we are in the process to optimize the visualization of Patronin by generating a red fluorescent protein tagged version, so that it can be combined with the GFP-based imaging marker. We are also in the process of generating imaging probes for Dynein, Spectrin and Shot, a putative linker protein between the microtubule and Spectrin systems. With respect to the imaging hardware, we have recently set up a super resolution spinning disk system that will improve on both fronts of imaging speed and resolution.
2. On the front of mechanical manipulation, we will continue our characterization of the loss-of-function phenotype of alpha-Spectrin. Design and implementation of methods for the perturbation and manipulation of components involved in apical dome mechanics are also underway.
3. On the integration of experiments and theory, we hope to begin performing quantitative analysis of components of the apical dome system so that the molecular dynamics model that Dr. Tarama has developed can start using realistic, measured parameters for simulation. In addition, In the next phase of model development, the membrane surface, currently modeled as a immobile, stiff boundary, will be allowed to deform. Finally, extension of the current 2D model into a 3D version will also be attempted.
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