High-Throughput, Multiplexing-Ready Intracellular Pressure Probes

高通量、可多重使用的细胞内压力探针

基本信息

批准号：
10431428
负责人：
Yun Chen
金额：
$ 26.88万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-17 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10431428
关键词：
3-Dimensional Actomyosin Address Anterior Calibration Cell Shape Cell Size Cell division Cell membrane Cell physiology Cells Cellular biology Coin Complex Contracts Cytoplasm Cytoskeleton DNA Data Development Electrodes Electroporation Embryo Energy Transfer Equilibrium Exhibits Extracellular Matrix Fibroblasts Fluorescence Resonance Energy Transfer Fostering Four-dimensional Heterogeneity Human Image Individual Lipid Bilayers Liposomes Maps Measurement Measures Mechanics Membrane Methods Microelectrodes Motion Nanotechnology Phenotype Physiological Positioning Attribute Process Reaction Time Research Resolution Rupture Shapes Signal Transduction Silicon Structure Subcellular Spaces Surface Technology Testing Time Vacuum Variant Water base biophysical properties cell behavior cell cortex cell motility cytotoxicity density design extracellular fluorophore insight macromolecule migration multiplexed imaging nanoforms nanosized novel performance tests pressure pressure sensor prevent programs rational design response scaffold sensor spatiotemporal stem submicron temporal measurement tool water channel

项目摘要

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.

项目概要众所周知，在单细胞水平上，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发生变化，并且压力的划分可能对于动态细胞功能至关重要，因此，有必要开发绘制异质图谱的方法。考虑到当前的技术限制，亚微米分辨率下单个细胞内的细胞内压力具体来说，研究细胞内压力如何调节细胞过程是一项挑战。作为细胞皮层的突出，异质且动态地产生某些表型，例如这一挑战源于缺乏与高通量兼容的纳米尺寸传感器。吞吐量多重成像，以便局部细胞内压力和其他动态过程可以在整个细胞上同时测量。在此，我们建议开发一种纳米形式的高通量、可多重使用的细胞内探针。大小的脂质体被基于 DNA 的支架包围，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脂质体的是弹性 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，这由于脂质体体积的压力依赖性变化而延伸或收缩。传感器，被称为“水通道蛋白脂质体压力传感器（ALPS）”，将被输送到细胞质当细胞质中的压力发生变化时，内化的 ALPS 将通过水流出来改变其体积。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。作为概念证明，我们将使用 ALPS 绘制单细胞内诱发的动态压力场图使用分区压力在 3D 矩阵内迁移；结果将与直接进行比较；通过 0.5 μm 微电极获得的测量结果具有有限的空间分辨率，这是当前最先进的技术。如果成功的话，我们将产生一种新的工具，以前所未有的方式测量细胞内压力时空分辨率，有望提供有关局部细胞内压力如何变化的见解当细胞在 3D 地形中导航时动态变化。