In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures of Xenopus laevis egg extracts and demembranated Xenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol:vesicle ratio of 10:1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol:vesicle ratio of 5:1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol:vesicle ratio of 2.5:1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cisternae, which were then stacked one on top of another.