Nsp1_CYT associates with NP and contributes to nuclear positioning. (A) Schematic of the association of Nsp1_CYT with ER tubules that connect the mother NE and daughter cortex through the exocyst complex and contribute to the formation of NP. Red ovals, NPCs; green triangles, Nsp1_CYT; grey square, exocyst complex. The arrow indicates NP dynamics. (B) Maximum-intensity projections of two time lapses (Δt is 3 min) of a deconvolved z series of fluorescence images. Left panels show the distribution of Nsp1-GFP (PCCPL393; Video 5) and right panels of Nup170-GFP (PCCPL392; Video 8) in cdc6-1 cells. Arrowheads point to an Nsp1-GFP_CYT focus that interacts with NP. Bar, 2 µm. (C) Nsp1-FRB-GFP/Hmg1-mCherry (PCCPL487)– or Nup192-FRB-GFP/Hmg1-mCherry (PCCPL552)–expressing cells were imaged in the presence of DMSO or rapamycin. Individual unbudded cells were followed by time-lapse microscopy until anaphase completion (∼2 h). The images shown are maximum-intensity projections of a z series of deconvolved images of cells at the time point just before and after anaphase. M and D, mother and daughter, respectively. The position of the bud neck is indicated by the arrows. Bars, 2 µm. (D) Quantification of C where the percentage of cells showing a mispositioned nucleus is plotted under DMSO and rapamycin conditions. 36 ≤ n ≤ 96. Error bars indicate the standard error of the mean.