PDF(Pubmed)
Abstract：
The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1\'s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.
摘要：
细胞微环境的微观几何形状深刻影响细胞行为，然而，它与普遍表达的机械敏感性离子通道PIEZO1之间的联系仍不清楚。在这里，我们描述了一种荧光微量移液管抽吸测定法，可以实时同时显示细胞内钙动力学和细胞骨架结构，在不同的微量移液管几何形状。通过将弹性壳有限元分析与荧光寿命成像显微镜相结合，并采用PIEZO1特异性转基因红细胞和HEK细胞系，我们证明了抽吸的微观几何形状与PIEZO1介导的钙信号之间的直接相关性。我们发现，微量移液管尖端角度和物理收缩的增加导致F-肌动蛋白的显着重组，积聚在抽吸的细胞颈部，并随后放大细胞圆顶处的张力应力以诱导更多的PIEZO1活性。F-肌动蛋白网络的破坏或其流动性的抑制导致PIEZO1介导的钙内流的显着下降，强调其在几何约束下的细胞机械传感中的关键作用。
