title: Ionophore-releasable lumenal Ca2+ 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 Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ 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 Ca2+ 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 Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.
authors.affiliation: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: I C
authors.initials: IC
authors.lastname: Marshall
authors.forename: T M
authors.initials: TM
authors.lastname: Gant
authors.forename: K L
authors.initials: KL
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002457:Cell Extracts; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology*; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000378:metabolism*; D056489:Nucleoplasmins; D010063:Ovum; D010750:Phosphoproteins; D019284:Thapsigargin / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D007476:Ionophores; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D000001:Calcimycin; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid