Assembly of higher order Nup133 structures is restricted to the nascent nuclear envelope membrane. (A) Association of Nup133, a component of the Nup107–160 complex, and Nup62 with the chromosome mass during early anaphase. The image is the middle section of a HeLa cell coexpressing GFP×3-Nup133 and H2B-mCherry (left) or GFP-Nup62 and H2B-mCherry (right) 30 s before its recruitment onto the nuclear envelope. The signal of H2B-mCherry (not depicted) was used to delineate the outer contours of the chromosome masses (dotted red lines). (B) Mean fluorescence intensity ratios ± SD of chromosome mass to cytosol for Nup133 and Nup62. The difference in ratios is statistically significant (P = 0.008). Chrom., chromosome. (C) A 2D time lapse from a mitotic HeLa cell expressing GFP×3-Nup133 and mCherry-Sec61β showing the recruitment of Nup133 onto the nuclear envelope (also see Video 6). The boxed regions are enlarged in D, and further enlarged boxed regions from D are shown in E. The onset of nuclear envelope formation is at 0 s. Open arrowheads show Nup133 associated with kinetochores. Closed arrowheads show Nup133 recruited to the nascent nuclear envelope. Note that Nup133 is absent on the surface of the chromosome mass at sites devoid of the nuclear envelope. Bars: (A and C) 10 µm; (D and E) 5 µm.