Fig. 2

lgd mutant MEs have a normal ILV content. (A–E) Ultra-structural analysis of lgd mutant and lgd cells rescued by additional genomic copies of shrub (lgd^d7; BAC^shrub). Representative TEM pictures of MVBs of wild-type (A), lgd^d7 mutant (B) and rescued lgd^d7 mutant (C) wing disc cells. (B”) As an example, colourized overlay of determined ILV area (46.9% of total MVB area) with the original electron microscopy image of the MVB is shown in (B’). Highlighted in bright red is the area which is not considered as ILV content/electron-dense material within the MVB. (D) Statistical analysis of MVBs of wild-type (blue), lgd mutant (red) and rescued lgd mutant (green) cells. The average area of lgd mutant MVBs is significantly increased in comparison to MVBs of wild-type cells. Furthermore, the enlargement of endosomes in lgd mutants is partially rescued in lgd mutant cells expressing two additional copies of BAC-shrub. (E) Pixelwise quantification of electron-dense material per area within the lumen of wt (blue) and lgd^d7 mutant (red) MVBs to measure ILV formation (as shown in B’, B”). A self-written macro for the image processing software “Fiji” was used (see the “Methods” section and [30]). Area quotients of several MVBs are collected and blotted in a box blot. The ILV content in lgd^d7 mutant is not changed in comparison to the wild-type [(D, E) wt n = 212 MVBs; lgd^d7 n = 230 MVBs; lgd^d7; BAC^shrub n = 277; (D) Kruskal-Wallis test, Dunn’s multiple comparison test p < 0.001 (***), p < 0.01 (**); (E) Mann-Whitney test, two-tailed; box plots: whiskers: 5–95 percentile; mean shown as “+”]. Scale bars (A–C) 250 nm