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bioRxiv · 10.1101/2024.06.25.600006

Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract

Takamori, S.·Mimura, H.·Osaki, T.·Kondo, T.·Shintomi, M.·Shintomi, K.·Ohsugi, M.·Takeuchi, S.
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Abstract

The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilize interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs.Characterization of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Our approach offers a versatile platform for constructing artificial cellular systems that closely mimic eukaryotic cells. TeaserA cell nucleus is reconstituted in lipid bilayer-enclosed confinements using egg extract from African clawed frogs.

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Takamori, S., Mimura, H., Osaki, T., Kondo, T., Shintomi, M., Shintomi, K., Ohsugi, M., Takeuchi, S.. 2024-06-25. Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract. https://doi.org/10.1101/2024.06.25.600006

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