| 研究課題/領域番号 |
19K06651
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
Phng LiKun 国立研究開発法人理化学研究所, 生命機能科学研究センター, チームリーダー (70794098)
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| 研究期間 (年度) |
2019-04-01 – 2022-03-31
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| キーワード | Endothelial cell / Angiogenesis / Actin cytoskeleton / Blebbing / Haemodynamic forces / Marcksl1 |
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| 研究実績の概要 |
The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in the zebrafish results in an increase and decrease, respectively, in EC size and the diameter of microvessels. Furthermore, endothelial overexpression of Marcksl1 induces ectopic blebbing on both apical and basal membranes, during and after lumen formation, that is suppressed by reduced blood flow. High resolution imaging of ECs in culture reveals that Marcksl1 promotes the formation of linear actin bundles and decreases actin density at the EC cortex. Additionally, the increase in the formation of actin bundles by the overexpression of the actin bundling protein Fascin1 in zebrafish ECs and the inhibition of branched actin formation by the drug CK666 both resulted in ectopic membrane blebbing in ECs. Our findings therefore demonstrate that a balanced network of linear and branched actin at the EC cortex is essential in conferring cortical integrity to resist the deforming forces of blood flow to regulate vessel structure.
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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
In this project, we have successfully generated tools (zebrafish transgenics and mutants) and methodologies (quantitative single cell analysis of endothelial cell shape and size in zebrafish and in cell cultures; quantitative analysis of actin dynamics and density in endothelial cortex etc.) to investigate the function of Marcksl1 in blood vessels. Importantly, we have unraveled the mechanism by which Marcksl1 regulates endothelial cell shape and vessel morphology. Findings from this funding are published in the paper "Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size" in Nature Communications in October 2020.
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| 今後の研究の推進方策 |
As previous methodology used to examine actin organization in zebrafish endothelial cells has been unsuccessful, we will next optimize imaging actin cytoskeleton in vivo by using a transgenic zebrafish actin reporter that labels F-actin brightly and super resolution imaging. We will also establish quantitative analysis of actin organization by quantifying actin bundle orientation, coherence, segment length and percolation density. Once established, we will use this methodology to investigate how actin binding proteins and changes in the perivascular environment affect actin cytoskeleton organization to regulate endothelial cell mechanics in vivo.
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| 次年度使用額が生じた理由 |
Because of the pandemic, funding anticipated for attending international conferences and visiting our collaborator was not used. I plan to use the remainder of the funds for zebrafish husbandry and purchase of experimental reagents and analyses tools necessary for performing quantitative analysis of actin cytoskeleton organization in wild-type and mutant zebrafish.
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