Inhibition of RanGTP-mediated spindle assembly pathway suppresses effects of CK2 inhibition in C. robusta eggs and X. laevis egg extracts. (A) Images of immunofluorescence of representative C. robusta oocytes treated for 1 h with DMSO, 250 μM CK2i, 250 µM importazole or 250 μM CK2i and 250 µM importazole, as indicated. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan). DMSO control spindle is image of the same spindle shown as experiment 3 DMSO control spindle in Fig. 2 A. CK2i spindle image is used as a representative image of EXTREME analysis category in Fig. 2 B and Fig. 5 B. (B) % of C. robusta oocytes displaying no (NONE), a limited (MILD), or an extensive (EXTREME) microtubule network around the spindle following treatment for 1 h with DMSO, 250 μM CK2i, 250 µM importazole or 250 μM CK2i and 250 µM importazole as indicated. Results from three independent experiments were pooled (n = 26 oocytes [DMSO], n = 29 oocytes [CK2i], n = 29 oocytes [importazole], n = 35 oocytes [CK2i + importazole]). Data shown for DMSO and CK2i are a subset of that shown in Fig. 2 B. Representative images of each analysis category shown are the same as representative images of each analysis category shown in Fig. 2 B. (C) Representative images of half spindles from spindle assembly reactions carried out in the presence of DMSO, 50 μM CK2i, 300 µM importazole or 50 μM CK2i and 300 µM importazole, as indicated. β-tubulin (magenta), DNA (cyan), TPX2 (green). (D) Representative images of half spindles from spindle assembly reactions carried out in the presence of DMSO and XB (control), 50 μM CK2i and XB (CK2i), 2.5 µM importin-β(71–876) and DMSO (Impβ[71–876]), or 50 μM CK2i and 2.5 µM importin-β(71–876) (CK2i + Impβ[71–876]), as indicated. β-tubulin (magenta), DNA (cyan), and TPX2 (green). (E) Violin plot of half spindle length of half spindles from experiment described in (C). n ≥ 324 half spindles per condition (from three cytoplasmic extracts [n ≥ 105 per extract for each condition]). (F and G) Violin plot of β-tubulin (F) or TPX2 (G) intensity at spindle poles of half spindles from experiment described in C. n ≥ 420 half spindles per condition (from three cytoplasmic extracts [n ≥ 133 per extract for each condition]). (H) Violin plot of half spindle length of half spindles from experiment described in D. n ≥ 313 half spindles per condition (from three cytoplasmic extracts [n ≥ 104 per extract for each condition]). (I and J) Violin plot of β-tubulin (I) or TPX2 (J) intensity at spindle poles of half spindles from experiment described in D. n ≥ 329 half spindles per condition (from three cytoplasmic extracts [n ≥ 92 per extract for each condition]). (K) Schematic illustrating how the Ran pathway of spindle assembly may be altered by the decrease in CK2 activity at fertilization. Microtubules (magenta), DNA (cyan), activated Ran pathway (orange), TPX2 and other Ran-regulated SAFs (green). Where the Ran pathway is highly active, SAFs are released from importins and drive localized microtubule nucleation and stabilization. In meiosis, Ran pathway activation is limited to a small region surrounding chromosomes. In mitosis, Ran pathway activation extends to a broader area, leading to increased localization of TPX2 and other Ran-regulated SAFs, including ELYS and augmin, at spindle poles, increasing spindle length and driving astral microtubule polymerization at spindle poles. Statistical significance was determined by two-tailed Mann–Whitney tests (**** = P < 0.00005). For violin plots, lines indicate the median and upper and lower quartiles.