Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.
Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.
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DOI：
10.1038/srep25502
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发表时间：
2016-05-05
影响因子：
4.6
通讯作者：
Hordijk PL
中科院分区：
文献类型：
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作者：
Reinhard NR;van Helden SF;Anthony EC;Yin T;Wu YI;Goedhart J;Gadella TW;Hordijk PL
内皮细胞排列在脉管系统中，对于调节血压、血管通透性、凝血以及白细胞和肿瘤细胞的跨内皮迁移非常重要。 RhoGTP 酶是一组控制内皮屏障功能的蛋白质。本研究重点关注三种同源 (>88%) RhoGTPase：RhoA、RhoB、RhoC，其中 RhoB 和 RhoC 的表征还很有限。通过 RhoGTPase mRNA 表达分析，我们确定 RhoC 在原代人内皮细胞中表达最高。基于现有的 RhoA FRET 传感器，我们开发了新的 RhoB/C FRET 传感器来表征其时空激活特性。我们发现所有这些 RhoGTPase 传感器都能对生理相关激动剂（例如凝血酶）做出反应，达到瞬时、局部 FRET 比率变化高达 200%。这些 RhoA/B/C FRET 传感器显示局部 GEF 和 GAP 活性，并揭示 RhoA/C 和 RhoB 之间的空间激活差异。最后，我们使用这些传感器来监测 RhoA/B/C 的 GEF 特异性差异激活。总之，本研究在现有的 FRET 传感器工具包中添加了高对比度 RhoB/C FRET 传感器，并揭示了内皮细胞和 RhoGTPase 细胞生物学的新见解。这使我们能够在原代人类细胞中以高时空分辨率研究这些密切相关的 RhoGTP 酶的激活和信号传导。
Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells.