E191A + K192A (EK) mutation in Num1 disrupts patch assembly function. (A) NUM1-GFP mCherry-TUB1 cells were imaged in the same field with num1^EK-GFP cells. (top) Single–focal plane image of GFP and mCherry-Tub1. (bottom) A color intensity map showing that NUM1-GFP cells, but not num1^EK-GFP, exhibit bright cortical GFP patches. A.U., arbitrary unit. Bar, 1 µm. (B) The percentage of cells in A exhibiting cortical GFP foci with intensities >18,000 (arbitrary units; n ≥ 237 cells for each strain). (C) Histograms of fluorescence intensity of individual Num1-GFP or Num1^EK-GFP cortical foci (n ≥ 239 foci for each construct from a single experiment). $x−$ denotes mean ± SD. The blue dashed line indicates the mean intensity of Num1-GFP foci. The mean value of Num1-GFP is different from that in Fig. 2 D because of nonidentical imaging conditions. (D) The percentage of binucleated cells in cultures grown at 12°C for 15 h (n ≥ 510 cells for each strain). (E) num1^EK cells exhibited defects in moving the spindle. (left) The percentage of spindles that crossed the bud neck during a 10-min video for HU-arrested NUM1 and num1^EK cells in the kar9Δ background (P < 0.0001 by t test; n ≥ 133 spindles for each strain from a single experiment). (right) Histograms of penetration distance (as defined in Fig. 3 E) during spindle oscillation. P = 0.001 by a t test (n ≥ 37 spindles for each strain from a single experiment). $x−$ denotes mean ± SD. The red dashed line indicates that spindles in num1^EK cells failed to move for a distance >2.9 µm. All error bars are standard error of proportion.