Currey Marilena L, Kandula Viswajit, Biggs Ronald, Marko John F, Stephens Andrew D
Biology Department, University of Massachusetts Amherst, Amherst, USA.
Department of Molecular Biosciences and Department of Physics & Astronomy, Northwestern University, Evanston, USA.
Cell Mol Bioeng. 2022 Sep 6;15(4):303-312. doi: 10.1007/s12195-022-00734-y. eCollection 2022 Aug.
Force measurements of the nucleus, the strongest organelle, have propelled the field of mechanobiology to understand the basic mechanical components of the nucleus and how these components properly support nuclear morphology and function. Micromanipulation force measurement provides separation of the relative roles of nuclear mechanical components chromatin and lamin A.
To provide access to this technique, we have developed a universal micromanipulation apparatus for inverted microscopes. We outline how to engineer and utilize this apparatus through dual micromanipulators, fashion and calibrate micropipettes, and flow systems to isolate a nucleus and provide force vs. extensions measurements. This force measurement approach provides the unique ability to measure the separate contributions of chromatin at short extensions and lamin A strain stiffening at long extensions. We then investigated the apparatus' controllable and programmable micromanipulators through compression, isolation, and extension in conjunction with fluorescence to develop new assays for nuclear mechanobiology.
Using this methodology, we provide the first rebuilding of the micromanipulation setup outside of its lab of origin and recapitulate many key findings including spring constant of the nucleus and strain stiffening across many cell types. Furthermore, we have developed new micromanipulation-based techniques to compress nuclei inducing nuclear deformation and/or rupture, track nuclear shape post-isolation, and fluorescence imaging during micromanipulation force measurements.
We provide the workflow to build and use a micromanipulation apparatus with any inverted microscope to perform nucleus isolation, force measurements, and various other biophysical techniques.
The online version contains supplementary material available at 10.1007/s12195-022-00734-y.
对细胞核（最强的细胞器）进行力的测量推动了力学生物学领域的发展，以了解细胞核的基本机械组成部分以及这些组成部分如何恰当地支持核形态和功能。显微操作力测量可区分核机械组成部分染色质和核纤层蛋白A的相对作用。
为了使这种技术得以应用，我们开发了一种适用于倒置显微镜的通用显微操作装置。我们概述了如何通过双显微操作器设计和使用该装置，制作和校准微量移液器以及流动系统，以分离细胞核并提供力与伸长量的测量。这种力测量方法具有独特的能力，能够测量短伸长量时染色质的单独贡献以及长伸长量时核纤层蛋白A的应变硬化。然后，我们通过结合荧光的压缩、分离和伸长操作，研究了该装置可控且可编程的显微操作器，以开发新的核力学生物学检测方法。
使用这种方法，我们首次在其原实验室之外重建了显微操作装置，并重现了许多关键发现，包括多种细胞类型的细胞核弹簧常数和应变硬化。此外，我们还开发了基于显微操作的新技术，用于压缩细胞核以诱导核变形和/或破裂，跟踪分离后的核形状，以及在显微操作力测量过程中进行荧光成像。
我们提供了使用任何倒置显微镜构建和使用显微操作装置的工作流程，以进行细胞核分离、力测量以及各种其他生物物理技术。
在线版本包含可在10.1007/s12195-022-00734-y获取的补充材料。
