title: An in vitro system to study nuclear envelope breakdown.
abstract: During mitosis in vertebrate cells, the nuclear compartment is completely disintegrated in the process of nuclear envelope breakdown (NEBD). NEBD comprises the disassembly of nuclear pore complexes, disintegration of the nuclear lamina, and the retraction of nuclear membranes into the endoplasmic reticulum. Deciphering of the mechanisms that underlie these dynamic changes requires the identification of the involved molecular components and appropriate experimental tools to define their mode of action. Here, we describe an in vitro, imaging-based experimental system, which recapitulates NEBD. In our assay, we induce NEBD on nuclei of semi-permeabilized HeLa cells expressing fluorescently tagged nuclear envelope (NE) marker proteins by addition of mitotic cell extract that is supplemented with fluorescently labeled dextran. Time-lapse confocal microscopy is used to monitor the fate of the selected NE marker protein, and loss of the NE permeability barrier is deduced by influx of the fluorescent dextran into the nucleus. This in vitro system provides a powerful tool to follow NEBD and to characterize factors required for the reorganization of the NE during mitosis.
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Joseph
authors.initials: J
authors.lastname: Marino
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Lysie
authors.initials: L
authors.lastname: Champion
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Wandke
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Horvath
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Monika I
authors.initials: MI
authors.lastname: Mayr
authors.affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kutay
mesh_terms: D045744:Cell Line, Tumor; D003911:Dextrans / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D018613:Microscopy, Confocal / Q000379:methods*; D008938:Mitosis; D009685:Nuclear Envelope / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism
chemical_list: D003911:Dextrans; D028861:Nuclear Pore Complex Proteins; D049452:Green Fluorescent Proteins
keywords: In vitro system; Microscopy; Mitosis; Nuclear envelope; Nuclear envelope breakdown; Nuclear membrane; Nuclear pore complex; Nucleoporin