Figure 7

Cold depolymerization of spermatocyte meiotic spindle microtubules removes spindle pole TACC3 with impacts on chromosome alignment, but recovery experiments show rescue of spindle pole TACC3, microtubule reassembly and chromosome realignment unless in the presence of 3.5% 1,6-hexanediol that blocks spindle pole TACC3 but not initial spindle pole microtubule polymerization. Panel 1: (A–C) control prometaphase-II spermatocyte showing bipolar spindle assembly (A, B: microtubules, red), aligning chromosomes (A: DNA, blue), and GFP CETN2 centrioles (A, C: green, arrowheads) with spindle pole TACC3 (B, C: blue, arrowheads). (C) insets: details, GFP CETN2 centrioles (green) and TACC3 (blue). (D–F) Prometaphase-I spermatocyte exposure to cold conditions for 15 min initiates spindle microtubule disassembly (D, E: red), misaligned chromosomes (D: DNA, blue), and loss of spindle pole TACC3 (E, F: blue, arrowheads) marked by GFP CETN2 centrioles (D, F: green; arrowheads). (F) Insets: details, GFP CETN2 centrioles (green) and TACC3 (blue). (G–I) A metaphase-II spermatocyte exposed for 15 min to cold conditions plus 15 min rescue in warm conditions shows bipolar spindle reassembly (G, H: microtubules, red) with kinetochore microtubules (H: red, arrows), realigning chromosomes (G: DNA, blue) and spindle pole TACC3 (H, I: blue, arrowheads) at the GFP CETN2 centrioles (G, I: green, arrowheads). Assembling spindle kinetochore microtubules do not label with TACC3 (H: blue, arrows). (I) insets: details, GFP CETN2 centrioles (green) and TACC3 (blue). (J–L) A metaphase-I spermatocyte exposed for 15 min to cold conditions plus 15 min rescue in warm conditions in the presence of 3.5% 1,6-hexanediol shows extensive cytoplasmic TACC3 without binding at the GFP CETN2 centrioles (J, L: green, arrowheads), significant microtubule assembly from both spindle poles (J, K: red) with kinetochore microtubules (K: red, arrows) and still misaligned chromosomes (J: DNA, blue). The reassembling kinetochore microtubules do not label with TACC3 (K: blue, arrows). (L) Insets: details, GFP CETN2 centrioles (green) and TACC3 (blue). Scale bars = 5 µm. Panel 2: graphic analysis of TACC3 spindle pole detection shows significant deviation from non-treated controls following cold exposure (**; p < 0.01147), cold with 15 min rescue (***; p < 0.006156), and cold with 15 min rescue in presence of 3.5% 1,6-hexanediol (***; p < 0.01419).