title: A time-lapse imaging assay to study nuclear envelope breakdown.
abstract: Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.
authors.affiliation: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA.
authors.forename: Sunita S
authors.initials: SS
authors.lastname: Shankaran
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Mackay
authors.forename: Katharine S
authors.initials: KS
authors.lastname: Ullman
mesh_terms: D000818:Animals; D002021:Buffers; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000302:isolation & purification; D004072:Digitonin / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure; D010539:Permeability; D059010:Single-Cell Analysis / Q000379:methods; D059008:Time-Lapse Imaging / Q000379:methods*; D014981:Xenopus
chemical_list: D002021:Buffers; D002457:Cell Extracts; D007202:Indicators and Reagents; D004072:Digitonin