A newly synthesized bud-directed pool of Nsp1. (A) Schematic of the NPC with major structural units and representative nups indicated in bold. ONM and INM, outer and inner nuclear membrane, respectively. (B) Schematic of the RITE cassette inserted in frame at the 3′ end of nup genes. Nup-mCherry fusions (old) are produced in cells. The addition of estradiol releases a Cre recombinase–EBD fusion that promotes loxP-mediated recombination, which leads to the replacement of the mCherry gene with a GFP ORF and the production of a nup-GFP (new) protein. (C–F) Cells expressing the indicated Nup-RITE fusions (PCCPL520, PCCPL522, PCCPL526, and WZCPL2) were incubated in the presence of estradiol, and both Nup-GFP and Nup-mCherry were imaged every 3 min through the indicated anaphase stages. Each fluorescence micrograph is a maximum-intensity projection of a deconvolved z series. Note the arrowhead in F showing a bud-localized focus of Nsp1 that does not colocalize with the NE until the last time point (see MERGE and Video 1). C1–F2 are plots (left) of a ratio of tf (green and red) in daughters (tf_d) versus mothers (tf_m). These ratios were then divided by a ratio of the surface area of the proportion of the dividing nucleus (through a middle z plane) found in either the daughter (sa_d) or mother (sa_m) to yield plots at right. 6 ≤ n ≤ 13. Error bars represent the standard deviation from the mean value. Bars, 2 µm.