Artificial disruption of the nuclear/spindle envelope in mitosis causes aberrant spindle formation and chromosome missegregation. (A–D) S2 cells expressing mRFP–α-tubulin and histone H2B-GFP. The nuclear/spindle envelope was consecutively disrupted by laser microsurgery (arrowheads). (A) Control cell progressing through mitosis. Laser microsurgery was performed at a random site in the cytoplasm shortly after NEB (arrowhead). (B) Disruption of the NE in prophase or the spindle envelope in early prometaphase (C) caused aberrant spindle formation and chromosome missegregation. (D) Chromosomes of the severed nucleus in a binucleated prophase cell condensed prematurely, and centrosome-nucleated MTs grew in the cytoplasm toward the cut region. NEB of the unsevered nucleus initiated only several minutes later, and MTs grew predominantly into the nuclear space. Time is given in minutes:seconds, relative to laser microsurgery. Bars, 10 µm. (E) Model: a membranous spindle envelope preserves different molecular crowding states between the cytoplasm and the spindle region during mitosis. Large assemblies such as membranous organelles are excluded from the spindle region. Small molecules like soluble tubulin or Mad2 equilibrate in all available spaces after NEB and accumulate in the less crowded spindle region. Large nuclear assemblies, such as Tpr, are retained in the spindle region.