High-Throughput, Multiplexing-Ready Intracellular Pressure Probes

项目摘要 众所周知，在单个细胞的水平上，细胞内压力控制运动性、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，压力的区域化 可能是动态细胞功能所必需的。因此，有必要开发方法来映射异质 在亚微米分辨率下的单个细胞内的细胞内压力 技术.具体来说，研究细胞内压力如何调节细胞过程是具有挑战性的，例如 作为细胞皮层的突起，不均匀地和动态地，导致某些表型，如 定向迁移这一挑战源于缺乏纳米尺寸的传感器，这些传感器可兼容高性能的传感器。 通量多路成像，使得局部细胞内压力和其他动态过程可以 在整个细胞中同时测量。 在此，我们建议开发一种高通量的，多路复用的细胞内探针的形式，纳米- 由基于DNA的支架包围的大小的脂质体，水通道蛋白分子分布在脂质双层中。 连接DNA支架和水通道蛋白包埋的脂质体的是弹性DNA系链，其缀合有 促进共振能量转移（FRET）供体和受体荧光团在规定的间距， 由于脂质体体积的压力依赖性变化而延伸或收缩。纳米级的压力 传感器，创造了“水通道蛋白-花边脂质体压力传感器（ALPS）"，将被交付到细胞质中， 数量。当细胞质中的压力变化时，内化的ALPS将通过水流出改变其体积 或通过水通道蛋白流入，而DNA支架稳定脂质体以防止塌陷或破裂。 作为概念验证，我们将使用ALPS绘制单细胞内诱导的动态压力场 使用分区压力在3D矩阵内迁移;将结果与直接 通过0.5 μm微电极以有限的空间分辨率获得的测量，当前的最新技术水平。如果 如果成功，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，这有望提供关于局部细胞内压力如何变化的见解 当细胞在3D地形中导航时，

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.