The RanGTP gradient generates an effector gradient of Kinesin-14s for preferential parallel MT cross-linking and sliding near the spindle poles. (A) Representative confocal fluorescence (CyPet, YPet, and MT) and lifetime (FLIM) images of spindles assembled with importin α-CyPet + nontagged XCTK2 + Ran (importin α-CyPet + 20 µM Ran), importin α-CyPet + YPet-XCTK2, or importin α-CyPet + YPet-XCTK2 + 20 µM Ran). Scale bar: 10 µm. (B) YPet fluorescence line scans of spindles assembled in A of importin α-CyPet with YPet-XCTK2 ± Ran normalized to percentage spindle length (25 bins) and graphed as the mean ± SEM (n = 60 YPet-XCTK2 and 97 YPet-XCTK2 + Ran spindles from four to five independent experiments). (C) Lifetime line scans of spindles imaged in A where lifetimes are represented as the amplitude averaged lifetime (τAV/AMP), and lifetimes per percentage of spindle length are graphed as the mean ± SEM (n = 68 importin α-CyPet + XCTK2 + Ran spindles, 27 importin α-CyPet + YPet-XCTK2, and 52 importin α-CyPet + YPet-XCTK2 + Ran spindles from four to five independent experiments). (D) XCTK2 cross-links and slides both antiparallel and parallel MTs near the chromatin where RanGTP is high and the association with importin α/β is low. Near the spindle poles where RanGTP is low, importin α/β can bind to the XCTK2 tail and can selectively inhibit XCTK2 antiparallel MT cross-linking. Thus, the RanGTP gradient sets up an opposing effector gradient of importin α/β association with XCTK2 that promotes pole focusing through preferential parallel MT cross-linking and sliding near the poles. (E) In cancer cells with centrosome amplification and high RanGTP, we propose that heightened RanGTP increases Kinesin-14 association to the spindle and that the RanGTP gradient biases Kinesin-14 localization toward the spindle poles, where reduced importin α/β association sets up an effector gradient of increased Kinesin-14 cross-linking and sliding activity that mediates centrosome clustering for cancer cell survival.
