Fig. 6: Loss of Katanin disrupts the non-centrosomal microtubule array and apical-medial actomyosin-mediated apical constriction.

a Schematic of the ‘degradFP’ tissue-specific-degradation system (as in ref. ³⁹): tissue-specific expression of an F-box/anti-GFP-nanobody fusion protein, degradFP, (using UAS/Gal4)⁷¹ leads to tissue-specific degradation of any endogenously GFP/YFP-tagged protein, in this case Katanin80-YFP. b–d Expressing degradFP using fkh-Gal4 in the salivary gland placode (Kat80YFP degradFP fkhG4; c–c″) leads to significant loss of Katanin80-YFP compared to control (Kat80YFP degradFP ctrl; b–b″). Cell outlines are marked by E-Cadherin (E-Cad). d Quantification of Katanin80-YFP depletion (Kat80YFP degradFP ctrl n = 33 embryos; Kat80YFP degradFP fkhG4 n = 34 embryos; shown are mean ± SD, statistical significance was determined by two-sided unpaired Mann–Whitney test as p < 0.0001). b″ and c″ are higher magnifications of the white boxes marked in b and c, respectively. e–g In placodes where Katanin80-YFP is degraded (f, f′), microtubules (green, labelled for acetylated α-tubulin) remain localised within the apical domain and in contact with centrosomes (magenta, labeled for Asl) compared to control (e, e′) where a non-centrosomal longitudinal array is formed. e′ and f′ are magnifications of the areas indicated in e and f by a white box. g Quantification of the effect shown in e, f; (Kat80YFP degradFP ctrl: 440 cells from 14 embryos; Kat80YFP degradFP fkhG4: 541 cells from 20 embryos; shown are mean ± SD, statistical significance was determined by two-sided unpaired Mann–Whitney test as p < 0.0001). h-h‴ Katanin80-YFP degradation (h′) leads to a loss of apical constriction compared to control (h), apical area of cells of example placodes are shown in a heat map. h″ Quantification of apical area distribution of placodal cells in control (ctrl) and Katanin depleted (degradFP fkhGal4) placodes at stage 11 showing the cumulative percentage of cells relative to apical area size. h‴ Percentage of cells in different size-bins [Kolmogorov-Smirnov two-sample test, p « 0.001 (***)]. 12 placodes were segmented and analysed for control and 13 for Katanin80-YFP depletion, the total number of cells traced was N(Kat80YFP degradFP ctrl)=1373, N(Kat80YFP degradFP fkhG4) = 1162. i–k In placodes where Katanin80-YFP is degraded (j, j′), apical-medial F-actin (green, labeled using phalloidin) is reduced compared to control (i, i′) where apical-medial actin is highly prevalent. i′ and j′ are magnifications of the areas indicated in i and j by a white box. k Quantification of loss of apical-medial F-actin (Kat80YFP degradFP ctrl: 924 cells from 17 embryos; Kat80YFP degradFP fkhG4: 919 cells from 15 embryos); shown are mean ± SD, statistical significance was determined by two-sided unpaired Mann–Whitney test as p < 0.0001. The salivary gland placode is indicated by a white dotted line and the invagination point, where present, by an asterisk.