Fig 5. Reducing htt expression increases Abl-FRET biosensor phosphorylation state during MB development.
(A) Schematic representation of an imaged MB in the brain. (B) The Abl-FRET biosensor is based on mammalian CRK protein scaffold with two additional fluorescent proteins (CFP and YFP). ABL kinase activity induces phosphorylation of UAS-Abl-FRET biosensor leading to its spatial rearrangement and increased FRET efficiency [59]. (C) Representative maximal projection of the YFP and FRET signals recorded in the MB lobes of adult flies using confocal microscopy. These are presented as examples of the kind of image data that goes into the FRET ratio calculation. Scale bar: 10μm. (D) FRET and YFP signals are recorded on adjacent 0.5 μm confocal planes along all the anterior-posterior axis of the α and β MB lobes in adult flies. Either wild-type UAS-Abl-FRET biosensor or a mutated form (UAS-Y➔F Abl-FRET) are expressed in the αβ MB neurons using the c739-GAL4 driver. In the UAS-Y➔F Abl-FRET biosensor, the tyrosine located in ABL target site (PYAQP) was replaced by a phenylalanine (PFAQP) impairing phosphorylation [42]. FRET efficiency is significantly reduced for UAS-Y➔F Abl-FRET relative to UAS-Abl-FRET biosensor for all confocal planes considered except for the nine most anterior and six most posterior planes. Two-tailed Mann-Whitney tests on non-Normally distributed data. Results are mean ± SEM with n ≥ 7 MB for each confocal plane. (E) Reducing htt expression increases FRET efficiency of UAS-Abl-FRET biosensor in third instar larval MB lobes. The UAS-Abl-FRET biosensor is expressed in MB neurons using the c739-GAL4 driver in control (+/+) and httint/+ flies. FRET efficiency is significantly increased in httint/+ flies for all confocal planes along the anterior-posterior axis except for the three located 3 to 4 μm from the initial confocal plane. Two-tailed Mann-Whitney tests on non-Normally distributed data. Results are mean ± SEM with n ≥ 13 MB for each confocal plane. (F) FRET efficiency is globally reduced in UAS-Y➔F Abl-FRET mutant versus UAS-Abl-FRET. FRET efficiency is averaged for all confocal planes and all along the anterior-posterior axis. Two-tailed Mann-Whitney test with non-Normally distributed data. Results are mean ± SEM with n ≥462; *** p<0.001. (G) FRET efficiency is globally increased when htt expression is reduced. FRET efficiency is averaged for all confocal planes and all along the anterior-posterior axis. Two-tailed Mann-Whitney test on non-Normally distributed data. Results are mean ± SEM with n ≥ 1055; *** p<0.001. (H) Abl mRNA expression is not changed in L3 brains following htt partial inactivation. Abl expression was assessed using RT-qPCR in L3 brains of httint /+ versus WT (+/+) male flies. Results show three independent biological replicates. Full genotypes: Genotypes: y w67c23 / Y; c739-GAL4/+; UAS-Abl-FRET/+. y w67c23 / Y; c739-GAL4/+; UAS-Y-F Abl-FRET/+. y w67c23 / Y; c739-GAL4/+; httint UAS-Abl-FRET/+.