Overexpression of Pac1 reduces the extent by which Num1_CC depletes plus end dynein. (A) Representative images of cells expressing mTurquoise2-Tub1, Dyn1-3mCherry, either Num1_CC or Num1_CC^LL/EE, and either overexpressing Pac1 or Kip2, as indicated. Because of the distorted spindle phenotype in Kip2-overexpressing cells, Spc110-Venus was used to mark SPBs. All cells were grown in galactose-containing media to induce overexpression of Pac1, Kip2, Num1_CC, or Num1_CC^LL/EE, as indicated. Each image is a maximum-intensity projection of a 2-µm Z-stack of wide-field images. For the top row (Pac1 overexpressed), arrows indicate plus end foci, and arrowheads indicate SPB foci. For the bottom row (Kip2 overexpressed), arrows indicate plus ends with or without foci. Bars, 2 µm. (B) The percentage of cells that exhibit plus end (red) or SPB (green) Dyn1-3mCherry foci is plotted for cells shown in A and for cells shown in Figs. 1 D and 2 B. Error bars represent the standard error of proportion (n ≥ 113 cells). (C) Extent by which Num1_CC overexpression reduced the frequency of observing dynein plus end foci compared with the respective PAC1 KIP2 isogenic parent strain overexpressing Num1_CC^LL/EE. Asterisks indicate a statistically significant percent decrease (see B for P values). (D) Box plot of fluorescence intensity values of plus end–associated Dyn1-3mCherry (n ≥ 31 foci). 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. (E) Extent by which Num1_CC overexpression reduced the number of dynein molecules (i.e., fluorescence intensity) at plus ends compared with the isogenic PAC1 KIP2 parent strain overexpressing Num1_CC^LL/EE. Asterisks indicate a statistically significant percent decrease (see D for P values). See also Fig. S5. WT, wild type.