Figure 5: dLRRK and hLRRK2 promote cap-dependent translation in postsynaptic muscles.
From: LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction
(a) Quantification of 5′-UTR luciferase reporter activity in response to co-transfection with either wild-type hLRRK2wt or kinase-dead hLRRK23XKD normalized to psiCHECK2 response in HEK293T. n=3 experiments, *P=0.043. See also Supplementary Table 2. (b) Western blot analysis of in vivo Fur1-5′-UTR-eGFP reporter expression when co-expressed with either dLRRK3KD (+/UAS-Fur1-5′-UTR-eGFP; UAS-dLRRK3KD/MHC-Gal4) or wild-type dLRRKwt (UAS-dLRRKwt/UAS-Fur1-5′-UTR-eGFP; +/MHC-Gal4). Left: Western blotting probed with (top) anti-GFP and (bottom) anti-Actin as loading control. Right: quantification of Fur1-5′-UTR-eGFP expression normalized to actin levels. n=3 for each. *P<0.05. (c) Muscle 6 expression of Fur1-5′-UTR-eGFP in control (UAS-Fur1-5′-UTR-eGFP/24B-GAL4) and dLRRK-overexpressing larva (UAS-dLRRK/+; 24B-GAL4/UAS-Fur1- 5′-UTR-eGFP). Scale bar, 100 μm. (d) Quantification of fluorescence intensity of muscles 6 and 7 normalized to muscle volume and expressed as a percentage of control. n=20 and 15 muscles, **P=0.001. (e) Muscle 4 NMJs stained with anti-Discs large (Dlg, red), anti-Fur1 (green) in control (MHC-Gal4/+) and dLRRK muscle overexpression larvae (+/UAS-dLRRK; MHC-Gal4/+). Scale bar, 10 μm. (f) Quantification of fluorescence intensity within Dlg area of genotypes in e. n=18 and 16 NMJs. *P=0.044. (g) Quantification of Fur1 mRNA expression by qPCR. n=3 technical replicates. Error bars represent s.e.m. Student’s t-test.
