Nuclear assembly in giant unilamellar vesicles encapsulatingXenopusegg extract Article Swipe
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· 2024
· Open Access
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· DOI: https://doi.org/10.1101/2024.06.25.600006
· OA: W4400056651
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. Teaser A cell nucleus is reconstituted in lipid bilayer-enclosed confinements using egg extract from African clawed frogs.
