HURP is an obstacle for KIF18A on K-fibers in metaphase cells.

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HURP is an obstacle for KIF18A on K-fibers in metaphase cells. HURP is an obstacle for KIF18A on K-fibers in metaphase cells. (A) Metaphase-arrested cells treated with KIF18A siRNA (and HURP siRNAs in marked conditions) and transfected with GFP-WT KIF18A or GFP-sNL1. Arrows indicate GFP-KIF18A accumulation near kinetochores (ACA, red). (B) Metaphase-arrested cells treated with KIF18A siRNA and transfected with mCherry-WT KIF18A, mCherry-sNL1, and/or GFP-HURP. Arrows mark the regions of greatest accumulation of mCherry-WT KIF18A. Shown in (A) and (B) is one z-slice through the middle of the spindle. (C) Quantification of KIF18A accumulation at central K-fiber ends under the conditions mentioned earlier. Line scans were performed, normalized, and aligned as in Fig 1, then each line was divided at the dotted line (−0.32 μm) and the area under the curve calculated for each segment (top). The ratio of tip (0 to −0.32 μm from kinetochore) to lattice (−0.32 to −0.768 μm) Kif18A fluorescent intensity is reported in the following graphs. Quantification of GFP-WT and GFP-sNL1 with HURP knockdown (middle). Bars are mean ± SD. *, adjusted P = 0.0135; ***, adjusted P = 0.0002 with 95% confidence interval by one-way ANOVA with Tukey's multiple comparisons test. Data obtained from two independent experiments with the following cell and line numbers: GFP-WT (12 cells, 31 lines), GFP-WT + HURP siRNA (7 cells, 11 lines), and GFP-sNL1 (14 cells, 50 lines). GFP-sNL1 + HURP siRNA (12 cells, 29 lines). Quantification of mCherry-WT and mCherry-sNL1 with GFP-HURP overexpression (bottom). ****, adjusted P < 0.001; n.s., not significant (adjusted P = 0.2061) with 95% confidence interval by one-way ANOVA with Tukey's multiple comparisons test. Data obtained from two independent experiments with the following cell numbers: mCh-WT (13 cells, 37 lines), mCh-WT + HURP-GFP (16 cells, 63 lines), mCh-sNL1 (12 cells, 48 lines), and mCh-sNL1 + GFP-HURP (16 cells, 51 lines). Source data are available for this figure. Heidi LH Malaby et al. LSA 2019;2:e201800169 © 2019 Malaby et al.