Inophore-releasable lumenal Ca 2+ stores are not required for nuclear envelope assembly or nuclear protein import in Xenopus egg extracts

Abstract

The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca 2+ stores via inositol 1,4,5-trisposphate-gated Ca 2+ channels is required for nuclear assembly in Xenopus egg extracts [1,2]. Other work suggested that lumenal Ca 2+ stores are required for nuclear protein import using vitro [3]. Here, we rigorously tested the role of lumenal Ca 2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca 2+ stores were depleted by pretreating the extracts with Ca 2+ ionophores (ionomycin, A23187) or inhibitors of Ca 2+-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca 2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca 2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca 2+-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca 2+ stores confirs a previous unpublished observation by Greber and Gerace [3], and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca 2+. Finally, we concluded that, contrary to our expectations, lumenal Ca 2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.