High-Throughput, Multiplexing-Ready Intracellular Pressure Probes

项目总结 众所周知，在单个细胞水平上，细胞内压力控制运动、形状、体积和增殖。 越来越多的证据表明，压力可以在单个细胞内变化，压力的分区 可能是动态细胞功能所必需的。因此，开发绘制异质地图的方法是至关重要的 考虑到电流的技术限制，单个细胞内的亚微米分辨率的细胞内压力 技术具体地说，研究细胞内压力如何调节细胞过程是具有挑战性的，例如 作为细胞皮质的突起，异质地和动态地导致某些表型，例如 定向迁移。这一挑战源于缺乏兼容于高性能传感器的纳米级传感器 吞吐量多路传输成像，以便局部细胞内压力和其他动态过程可以 同时在整个细胞内测量。 在这里，我们建议开发一种高通量、多路传输就绪的纳米形式的细胞内探针。 大小的脂质体包裹在基于DNA的支架上，水通道蛋白分子分布在脂双层中。 连接DNA支架和包埋水通道蛋白脂质体的是连接有弹性的DNA系链 以规定的间距培养共振能量转移(FRET)供体和受体荧光团，这 由于脂质体体积随压力变化而伸展或收缩。纳米尺寸的压力 传感器，被称为“水通道蛋白镶嵌脂质体压力传感器(Alps)”，将被输送到细胞质中。 数量。当细胞质内的压力变化时，内化的阿尔卑斯山会通过水的外流改变其体积。 或通过水通道流入，而DNA支架稳定脂质体以防止崩溃或破裂。 作为概念验证，我们将使用Alps来映射单个细胞内诱导的动态压力场 使用分区压力在3D矩阵内迁移；结果将与直接 用空间分辨率有限的0.5-μm微电极获得的测量，是目前最先进的。如果 成功后，我们将产生一种前所未有的测量细胞内压力的新工具 时空分辨率，承诺提供关于局部细胞内压力如何变化的洞察力 像单元格一样动态地在3D地形中导航。

PROJECT SUMMARY It is known that at the level of a single cell, intracellular pressure governs motility, shape, volume, and proliferation. Mounting evidence suggests that pressure can vary within a single cell and the compartmentalization of pressure may be essential for dynamic cell function. Thus, it is essential to develop approaches to map the heterogeneous intracellular pressure within a single cell at submicron resolution given the technical limitations of the current technology. Specifically, it is challenging to study how intracellular pressure regulates cellular processes, such as protrusion of the cell cortex, heterogeneously and dynamically, to result in certain phenotypes, such as directional migration. This challenge stems from the lack of nano-sized sensors that are compatible for high- throughput multiplexing imaging so that local intracellular pressure and other dynamic processes can be simultaneously measured across the cell. Herein we propose to develop a high-throughput, multiplexing-ready intracellular probe in the form of nano- sized liposomes enclosed by DNA-based scaffold with aquaporin molecules distributed in the lipid bilayer. Joining the DNA scaffold and the aquaporin-embedded liposome are elastic DNA tethers conjugated with Foster Resonance Energy Transfer (FRET) donor and acceptor fluorophores at prescribed spacing, which extend or contract as the result of pressure-dependent changes to liposome volume. The nano-sized pressure sensor, coined “aquaporin-laced liposome pressure sensor (ALPS)”, will be delivered to the cytoplasm in quantity. Upon pressure changes in the cytoplasm, the internalized ALPS will change its volume by water efflux or influx through the aquaporin, while the DNA scaffold stabilizes the liposome to prevent collapse or rupture. As a proof of concept, we will then use ALPS to map the dynamic pressure field induced within single cells using compartmentalized pressure to migrate within 3D matrix; the results will be compared to the direct measurements obtained by 0.5-μm micro-electrodes with limited spatial resolution, the current state-of-art. If successful, we will generate a novel tool for measuring intracellular pressure with unprecedented spatiotemporal resolution, which promises to provide insights on how local intracellular pressure changes dynamically as cells navigate the 3D terrain.