Figure 5

Unlike TACC3, GFP CETN2-expressing spermatocyte spindle pole cKAP5/chTOG is not entirely removed after exposure to MLN 8237 Aurora A kinase inhibitor despite spindle microtubule disorganization and chromosome misalignment. Panel 1: (A–C) Control GFP-CETN2-expressing spermatocytes (A, B: green) with spindle pole cKAP5/chTOG (B, C: blue) on a bipolar spindle (A, C: red, microtubules; A: blue, DNA). (D–F) 500 nM MLN 8237 (30-min) reduced visible spindle pole cKAP5/chTOG (E, F: blue; insets, details) with increased intra-spindle cKAP5/chTOG detection, though not on abnormal assembled spindle microtubules (D, F: red, microtubules; D: blue, DNA). (G–I) 500 nM MLN 8237 rescue experiments (30-min) showed spindle pole cKAP5/chTOG recovery (H, I: blue; insets, details) at the GFP CETN2-expressing centrioles (G, H: green) as spindle kinetochore microtubules reassembled (G, I: red, arrows) but chromosome remained unaligned (G: DNA, blue). Panel 2: cKAP5/chTOG relative fluorescent intensity (left) and surface intensity plots (right) for control (upper panels), 500 nM MLN 8237 exposed (middle panels) and 500 nM MLN 8237 with a 30 min rescue (lower panels). Control spermatocytes show mostly spindle pole cKAP5/chTOG intensity. MLN 8237 exposure reduces spindle pole cKAP5/chTOG and increased intra-spindle cKAP5/chTOG intensities. Rescue experiments partially re-establishes spindle pole cKAP5/chTOG while reducing intra-spindle intensities. Surface intensity plots: GFP CETN2 centriole plot (green) overlaid on cKAP5/chTOG plot (grey). Panel 3: images for intensity measurements shown in panel 2. Control (A–D), 500 nM MLN 8237 exposed (E–H) and rescue experiments from 500 nM MLN 8237 treatment (I–L). (A, E, I) bars, pole-to-pole measurements for intensity plotting. (D, H, L) overlays of cKAP5/chTOG (blue), microtubules (red) and GFP-CETN2 centrioles (green). MLN 8237 exposure disrupts normal bipolar spindle configuration (F; red, microtubules), reduces spindle length (G: green, GFP CETN2 centrioles), and increases cKAP5/chTOG (E: blue) within the spindle but not binding on remaining microtubules (H: overlay). Rescue experiments partially reverses spindle pole cKAP5/chTOG intensity while reducing intra-spindle protein intensity. All images: GFP CETN2-expressing centrioles (green) immunolabeled for YL1/2 microtubules (red), cKAP5/chTOG (blue), except panel 1 (A, D, G) (blue, DNA). Scale bars = 5 μm.