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. 2020 Jul 1;219(8):e201907016. doi: 10.1083/jcb.201907016

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© 2020 Sofroni et al.

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Figure 5. — Analysis of meiotic progression in WT and VF cdkd;3 mutants. (A) Confocal laser scanning micrographs of male meiocytes expressing TagRFP:TUA5 (magenta) and GFP:SYP132 (green) during meiotic progression in WT (time indicated with white numbers: h:min). See also Video 1 A. (B) Meiotic progression in VF cdka;1/− cdkd;3/+ (time indicated with white numbers: h:min) showing the conversion of a simultaneous into a successive cytokinesis in one male meiocyte highlighted with a green circle (from Video 2). TagRFP:TUA5 in magenta and bright field in gray, overlay in the third row, cell wall deposition is marked with red arrows. (C) Meiotic progression in one meiocyte (green circle) of VF cdka;1/− cdkd;3/− mutant expressing TagRFP:TUA5 (in magenta) and the respective overlay with the bright field in the second row, showing microtubule dynamics and cell wall deposition (red arrows) from late prophase I to meiotic exit (from Video 3; time indicated with white numbers: h:min). (D) Comparison of simultaneous cytokinesis in WT versus meiotic exit in VF cdka;1/− cdkd;3/− using GFP:SYP132 (green) as plasma membrane marker (from Video 4). Red asterisks mark the initiation and the outside-in direction of cell wall deposition during cytokinesis; time indicated with white numbers: h:min). (E–H) Comparison of the duration of meiotic stages for single meiocytes between WT (E), VF cdka;1/− (F), VF cdka;1/− cdkd;3/+ (G), and VF cdka;1/− cdkd;3/− (H). Every line represents a single cell undergoing meiosis, and every square, a 10-min interval of a specific meiotic stage: diakinesis (D), metaphase I (M1), interkinesis (I), metaphase II (M2), and telophase II/tetrad (T). In some mutants, exit after interkinesis (E) or a first cytokinesis (FC) is observed. After the second meiotic division, a second cytokinesis (SC) finally leads to the formation of meiotic products. Data aligned by taking a starting point 10 min before the first meiotic spindle is visible and, as final time point, 10 min after the spindle of the second meiotic division disappears. (I) Box plots of metaphase I, interkinesis, and metaphase II duration in WT (n = 38), VF cdka;1/− (n = 40), VF cdka;1/− cdkd;3/+ (n = 34), and VF cdka;1/− cdkd;3/− (n = 37). Red dots represent the mean value. Level of significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) determined by one-way ANOVA followed by Tukey’s test. (J) Cartoons summarizing microtubule organization during meiotic progression in WT versus VF cdka;1 cdkd;3 mutant combinations. Green arrows represent the direction of microtubule removal in the midzone during late interkinesis. Scale bar in A–D, 10 µm.