Overexpression of Num1_CC depletes Pac1 from plus ends and disrupts dynein-Pac1 interaction. (A) Representative images of GAL1p:num1_CC cells expressing mTurquoise2-Tub1, Pac1-3mCherry, and Dyn1-3YFP used for quantitation in B, C, and E. The arrow indicates the plus end focus, and arrowheads indicate SPB foci. (B) The percentage of cells that exhibit plus end (red) or SPB (green) fluorescent Pac1-3mCherry foci is plotted for the cells shown in A. Error bars represent the standard error of proportion (n ≥ 122 cells). (C) Box plot of fluorescence intensity values of plus end–associated Pac1-3mCherry (n ≥ 26 foci). (D) Western blot of Pac1-13myc–expressing GAL1p:num1_CC or GAL1p:num1_CC^LL/EE cells (as indicated) grown in galactose-containing media with loading control (anti–α-tubulin). (E) The extent of Pac1-3mCherry and Dyn1-3YFP colocalization is plotted for the indicated cells (n ≥ 61 fluorescent foci). (F) Representative images of GAL1p:PH-DYN1-3mCherry cells expressing Pac1-3YFP and either Num1_CC or Num1_CC^LL/EE. Arrowheads indicate cortical foci. (G and I) The percentage of cortical PH-Dyn1-3mCherry foci that colocalize with either Pac1-3YFP (n ≥ 49 foci; G) or Jnm1-3YFP (n ≥ 55 foci; I) is plotted for cells expressing either Num1_CC or Num1_CC^LL/EE. (H and J) Box plot of fluorescence intensity values for either cortical Pac1-3YFP (n ≥ 25 foci; H) or Jnm1-3YFP foci (n ≥ 44 foci; one outlier was omitted from the plot for display purposes only; J). For all box plots, the whiskers define the range of data, boxes encompass the 25th to 75th quartiles, the line depicts the median value, and the “x” depicts the mean value. All images are maximum-intensity projections of a 2-µm Z-stack of wide-field images. Bars, 2 µm. See also Fig. S4. WT, wild type.