School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC 3083, Australia.
School of Engineering, RMIT University, Melbourne, VIC 3001, Australia.
Sci Rep. 2017 Nov 21;7(1):15942. doi: 10.1038/s41598-017-16276-7.
Localized Ca influx via TRPV4 on the surface of endothelial cells greatly influences endothelial adaptation to blood flow, but how mechanical stress from blood flow controls TRPV4 integration into this physiological function is not fully understood. Here, we studied the spatial organization of TRPV4 and its relationship to the adherens junction component β-catenin using single- and dual-color direct stochastic optical reconstruction microscopy (dSTORM). In non-stimulated endothelial cells, TRPV4 is clustered in small protein islands, as is β-catenin. Using dual-color imaging, we found that TRPV4 and β-catenin reside in similar islands and can be found at both the basolateral and basal membranes. Following shear stress stimulation, TRPV4 molecules formed smaller clusters, with the majority residing outside of clusters. Further shear stress stimulation changed the molecular distribution of TRPV4 molecules, limiting them to the basal membrane. This redistribution and the smaller clusters resulted in the segregation of TRPV4 from β-catenin. Furthermore, TRPV4 trafficking was controlled by focal adhesion kinase and activation of the α5ß1 integrin. These highly differentiated spatial redistributions suggest that mechanotransduction of blood flow is controlled via a more complex hierarchy than previously thought.
局部 Ca2+内流通过血管内皮细胞表面的 TRPV4 极大地影响了内皮细胞对血流的适应，但血流产生的机械应力如何控制 TRPV4 整合到这种生理功能中尚不完全清楚。在这里，我们使用单重和双重颜色直接随机光学重建显微镜（dSTORM）研究了 TRPV4 的空间组织及其与黏着连接成分β-连环蛋白的关系。在未受刺激的血管内皮细胞中，TRPV4 与β-连环蛋白一样，聚集在小的蛋白岛中。通过双重成像，我们发现 TRPV4 和 β-连环蛋白位于相似的岛中，并且可以在基底外侧和基底膜上找到。在受到剪切力刺激后，TRPV4 分子形成较小的簇，其中大多数位于簇外。进一步的剪切力刺激改变了 TRPV4 分子的分子分布，将其限制在基底膜上。这种重分布和较小的簇导致 TRPV4 与β-连环蛋白的分离。此外，TRPV4 的运输受粘着斑激酶和α5β1 整合素的激活控制。这些高度分化的空间重分布表明，血流的机械转导受到比以前认为的更复杂的层次控制。
