Figure 8.
Comparison of PLP and KHC loss of function in NBs. (A i–iii) Schematic of centrosome asymmetry in a wild-type NB. (i) Mother centriole (pink) sheds PCM (green). (ii) Mother centriole migrates to the basal side. (iii) Mother centriole recruits PCM in the following prophase. (B) Schematic showing the main phenotypes in plp− mutants: Centrioles do not migrate away from the apical domain, mother centriole retains PCM, some NBs inherit supernumerary centrioles. (C–E) Live imaging of NBs expressing the centriole marker mNG::SAS-4 (cyan, arrows) and the MT marker Jupiter::mCherry (red). The apical daughter centriole is indicated in control NB (arrow), metaphase spindle axis is indicated by yellow line. Unlike controls (C), centrioles do not migrate away from the apical cortex following PLP (D) or KHC (E) knockdown. (F) The angle between the two centrosomes is significantly reduced at prophase following PLP or KHC knockdown (Control angle: 137 ± 25, n = 34. PLPRNAi angle: 84.98 ± 39, n = 62. KHC RNAi angle: 88.94 ± 40, n = 52; data = mean ± SD; ANOVA P = <0.0001, Tukey multiple comparison: P = <0.0001 between Control and RNAi conditions), ns = not significant. (G) The time taken for one centriole to cross the cell midline (equator) is significantly increased following PLP or KHC knockdown compared to controls (Control separation time: 26.43 ± 8, n = 28. PLPRNAi separation time: 55.65 ± 29.7, n = 54. KHCRNAi separation time: 63.84 ± 24.4, n = 43; data = mean ± SD; ANOVA P = <0.0001, Tukey multiple comparison: P = 0.001 between Control and RNAi conditions). (H) Representative NBs showing supernumerary centrioles are present in plp− (plp2171/df(3L) Brd15, 10%, n = 258 NBs, four brains) mutants but not in control (y,w, 1.1%, n = 229 NBs, four brains) or khc− (khc8/khc63, 1.15%, n = 195 NBs, four brains) mutants. Numbers represent percentage of cells carrying >2 centrioles as determined by Asl puncta (magenta) (I) Time series showing supernumerary centrioles following plp− loss of function arise from a failure of centrosomes to separate in prophase. (J) Fixed neuroblasts showing Gamma-Tubulin (magenta) associates with both centrioles (green) following PLP or KHC knockdown. (K) Quantification of the asymmetric index showing a significant reduction in gamma tubulin asymmetry in PLP or KHC knockdown (Control ASI: 0.7 ± 0.29, n = 37. PLPRNAi ASI: 0.49 ± 0.33, n = 34. KHCRNAi ASI: 0.5 ± 0.37, n = 46; ANOVA: P = <0.0001, Tukey pairwise comparison: P = <0.0001 between Control and all other conditions). Data = mean ± SD. Scale bars: 5 µm.

Comparison of PLP and KHC loss of function in NBs. (A i–iii) Schematic of centrosome asymmetry in a wild-type NB. (i) Mother centriole (pink) sheds PCM (green). (ii) Mother centriole migrates to the basal side. (iii) Mother centriole recruits PCM in the following prophase. (B) Schematic showing the main phenotypes in plp− mutants: Centrioles do not migrate away from the apical domain, mother centriole retains PCM, some NBs inherit supernumerary centrioles. (C–E) Live imaging of NBs expressing the centriole marker mNG::SAS-4 (cyan, arrows) and the MT marker Jupiter::mCherry (red). The apical daughter centriole is indicated in control NB (arrow), metaphase spindle axis is indicated by yellow line. Unlike controls (C), centrioles do not migrate away from the apical cortex following PLP (D) or KHC (E) knockdown. (F) The angle between the two centrosomes is significantly reduced at prophase following PLP or KHC knockdown (Control angle: 137 ± 25, n = 34. PLPRNAi angle: 84.98 ± 39, n = 62. KHC RNAi angle: 88.94 ± 40, n = 52; data = mean ± SD; ANOVA P = <0.0001, Tukey multiple comparison: P = <0.0001 between Control and RNAi conditions), ns = not significant. (G) The time taken for one centriole to cross the cell midline (equator) is significantly increased following PLP or KHC knockdown compared to controls (Control separation time: 26.43 ± 8, n = 28. PLPRNAi separation time: 55.65 ± 29.7, n = 54. KHCRNAi separation time: 63.84 ± 24.4, n = 43; data = mean ± SD; ANOVA P = <0.0001, Tukey multiple comparison: P = 0.001 between Control and RNAi conditions). (H) Representative NBs showing supernumerary centrioles are present in plp− (plp2171/df(3L) Brd15, 10%, n = 258 NBs, four brains) mutants but not in control (y,w, 1.1%, n = 229 NBs, four brains) or khc− (khc8/khc63, 1.15%, n = 195 NBs, four brains) mutants. Numbers represent percentage of cells carrying >2 centrioles as determined by Asl puncta (magenta) (I) Time series showing supernumerary centrioles following plp− loss of function arise from a failure of centrosomes to separate in prophase. (J) Fixed neuroblasts showing Gamma-Tubulin (magenta) associates with both centrioles (green) following PLP or KHC knockdown. (K) Quantification of the asymmetric index showing a significant reduction in gamma tubulin asymmetry in PLP or KHC knockdown (Control ASI: 0.7 ± 0.29, n = 37. PLPRNAi ASI: 0.49 ± 0.33, n = 34. KHCRNAi ASI: 0.5 ± 0.37, n = 46; ANOVA: P = <0.0001, Tukey pairwise comparison: P = <0.0001 between Control and all other conditions). Data = mean ± SD. Scale bars: 5 µm.

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