Figure 4.

Drosophila, dAtad3^R472C is highly deleterious and increases membrane-bound cholesterol. dAtad3^R472C was expressed in Drosophila using the UAS-Gal4 system. (A) UAS-dAtad3^R472C expressed under different Gal4 drivers led to lethality, except with GMR-Gal4 (late onset eye and neuronal driver), insc-Gal4 (neuroblast driver) and ey-Gal4 (eye discs)⁵⁶ drivers, similar to that produced when expressing dAtad3 RNAi (UAS-dAtad3RNAi). (B) Light microscope images of the abnormal eye phenotypes of flies expressing dAtad3^R472C under the ey-Gal4 driver seen in ∼33% of the viable progeny, compared to the normal eyes of flies expressing no transgene (UAS-empty), or wild-type transgenic Atad3 (UAS-dAtad3WT). (C) Confocal micrographs of Drosophila larvae brain lobes carrying insc-Gal4 and UAS-mKate-D4 together with UAS-empty [control (i) or UAS-dAtad3^R472C (ii)]. Neuroblasts are labelled Miranda (blue) and ganglion mother cells (GMCs) by Prospero (green). mKate-D4 (red) is a membrane-bound cholesterol reporter. Scale bar = 50 µm. The cartoon represents the cholesterol distribution in membranes and its predicted accumulation and aggregation based on C(ii).