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利用活性微柱阵列测量细胞骨架的力学波动和流变学性质。

Measuring Cytoskeletal Mechanical Fluctuations and Rheology with Active Micropost Arrays.

机构信息

Department of Physics and Astronomy, Johns Hopkins University, Baltimore, Maryland.

Current address: Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina.

出版信息

Curr Protoc. 2022 May;2(5):e433. doi: 10.1002/cpz1.433.

DOI:10.1002/cpz1.433
PMID:35612274
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9321978/
Abstract

The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts' motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well-defined positions. Application of these high-precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special-purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of magnetic micropost arrays Basic Protocol 2: Data acquisition for cellular force fluctuations on non-magnetic micropost arrays Basic Protocol 3: Data acquisition for local cellular rheology measurements with magnetic microposts Basic Protocol 4: Data reduction: determining microposts' motion Basic Protocol 5: Data analysis: determining local rheology from magnetic microposts Basic Protocol 6: Data analysis for force fluctuation measurements Support Protocol 1: Fabrication of magnetic Ni nanowires by electrodeposition Support Protocol 2: Configuring Streampix for magnetic rheology measurements.

摘要

细胞肌动球蛋白细胞骨架的动力学对细胞功能的许多方面都至关重要。在这里，我们描述了使用主动微柱阵列探测器（AMPAD）来测量细胞骨架流变学和机械力波动的技术。AMPADS 是由柔性聚二甲基硅氧烷（PDMS）微柱阵列组成，其中一些微柱中嵌入了磁性纳米线，以便通过外部施加的磁场来驱动这些微柱的运动。本文介绍了以纳米级精度跟踪磁性微柱运动的技术，其速度高达每秒 100 个视频帧，以测量在明确定义位置处的局部细胞流变学。将这些高精度跟踪技术应用于与细胞接触的整个微柱阵列，还可以以高时空分辨率绘制细胞骨架机械波动动力学图。本文描述了（1）磁性微柱阵列的制作，（2）局部流变学和细胞骨架力波动映射的测量方案，以及（3）用于减少和分析这些数据的专用软件例程。 © 2022 作者。Wiley Periodicals LLC 出版的《当代协议》。 基本方案 1：磁性微柱阵列的制作 基本方案 2：非磁性微柱阵列上细胞力波动的数据采集 基本方案 3：使用磁性微柱进行局部细胞流变学测量的数据采集 基本方案 4：数据减少：确定微柱的运动 基本方案 5：数据分析：从磁性微柱确定局部流变学 基本方案 6：力波动测量数据分析 支持方案 1：通过电沉积制备磁性 Ni 纳米线 支持方案 2：为磁性流变学测量配置 Streampix。

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