Importin-β excess hinders RANGAP1 recruitment at KTs in metaphase cells. (A) RANGAP1 localization in metaphase cells transfected with vector or with importin-β derivatives. H2B-GFP was cotransfected to visualize chromosomes. Bar, 10 µm. (B) Frequency of metaphase cells that lack RANGAP1 at KTs (320–450 scored metaphases per construct, 4 experiments). **, highly significant differences between cells transfected with importin-β 1–876 or 45–462 compared with controls (P < 0.001, χ2 test); there is no statistical difference between importin-β I178A/Y255A and controls. (C) Quantitative analysis of RANGAP1 recruitment to KTs in metaphase cells transfected with H2B-GFP and either importin-β 1–876, or 45–462, or vector. Transfected cells were identified for their green fluorescent chromosomes (not depicted), and RANGAP1-specific fluorescence signals were measured at single CREST-stained KTs. For each sample, the histograms represent the frequency (expressed as %) of KTs in the indicated classes of RANGAP1-specific fluorescence (n: 207 KTs in 6 independent counts for control samples, 236 KTs in 7 counts for importin-β–transfected samples, and 250 KTs in 8 counts for importin-β 45–462-transfected samples). KT-associated RANGAP1 signals range in classes of lower intensity values in importin-β 1–876- or 45–462-transfected cells compared with controls. (D) Examples of KTs in which RANGAP1-specific fluorescence was measured. Bar, 2 µm.
