Enhanced spindle MT bundling in KIRC-2 and -3 cells affects Kif15 partitioning. (A) Kif15 mislocalizes to non–K-MTs in KIRC-2 and -3 cells. Maximum intensity z-projections of indicated cell types 2 d after transfection with nontargeting (Control) or Nuf2-targeting (-Nuf2) siRNAs and stained with antibodies targeting Kif15 (grayscale and red) and tubulin (green). DNA, blue. LUTs for grayscale and red channels are scaled identically. Bar, 10 µm. (B) Kif15 protein levels are not elevated in KIRC-2 or -3. Western blot of WCLs prepared from indicated cell types probed with antibodies targeting Kif15 (αKif15) and tubulin (DM1α). (C) Quantitation of B. Values represent the levels of Kif15 protein in indicated cell types normalized to the parental HeLa line. n = 2. Error bars, ± SEM; *, P ≤ 0.1 relative to parental HeLa line. (D) Cellular Kif15 primarily organizes into dimers. Histogram showing initial single-molecule fluorescence intensities of EGFP-Kif15 (red) from HeLa cell extracts. XMAP215-EGFP (green) represents monomer control. EGFP-HSET (blue) represents dimer control. Intensities are indicated in au × 10−3. n ≥ 1945 particles from ≥ 3 fields of view. Single Gaussian fits of fluorescence intensity distributions are overlayed. XMAP215-EGFP (green, R2 = 0.96), EGFP-HSET (blue, R2 = 0.90), and EGFP-Kif15 (red, R2 = 0.93). (E) Example traces showing photobleaching steps of single EGFP-Kif15 molecules from HeLa cell extracts. XMAP215-EGFP (green) represents monomer control. EGFP-HSET (blue) represents dimer control. Two example traces are shown for EGFP-Kif15 (red, cyan) highlighting the variability observed in photobleaching steps. Fluorescence intensities are indicated in au × 10−3, and the baseline is set to the background fluorescence. See also Fig. S3.
