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A Versatile Micromanipulation Apparatus for Biophysical Assays of the Cell Nucleus
INTRO: 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 separatio...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9474788/ https://www.ncbi.nlm.nih.gov/pubmed/36119136 http://dx.doi.org/10.1007/s12195-022-00734-y |
Sumario: | INTRO: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-022-00734-y. |
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