(A) Dorsal view of a whole-mount 10-day-old female
Drosophila brain (yw) stained with
anti-Fur1 (green).
(B–D) Left: adult Drosophila brain stained with
anti-TH (red) (B), anti-Fur1 (green) (C), and merged (D). Right: magnification
of the PPL1 cluster in boxed region for (B)–(D).
(A-D) Imaging was performed at 63x magnification. Thirty-six images were
captured and tiled following a rectangular grid mode using Zen software.
(E–G) PPL1 clusters double stained with anti-TH (red) and
anti-Fur1 (green) in flies expressing (E) UAS-LacZ (Ddc-Gal4/UAS-LacZ), (F)
UAS-LRRK2IT (Ddc-Gal4/+; UAS-LRRK2IT/+) and (G)
UAS-S6KSTDE (Ddc-Gal4/+; UAS-S6KSTDE/+) in
dopaminergic (DA) neurons.
(H) Quantification of Fur1 sensor fluorescence intensity relative to TH
for genotypes in (E)–(G). Each data point represents fluorescence from a
single neuron. n = 100 neurons from 10 PPL1 clusters for each genotype. Data are
represented as mean ± SEM. One-way ANOVA with Bonferroni post-test.
(I–L) PPM1-2 clusters expressing Fur1 sensor (5′UTR of
Fur1 upstream of EGFP) (green) and stained with anti-TH (red) in flies
co-expressing (I) UAS-LacZ (UAS-Fur1-sensor/UAS-LacZ; Ddc-Gal4/+), (J)
UAS-LRRK2IT (UAS-Fur1-sensor/+; Ddc-Gal4/UAS-LRRK2IT),
(K) UAS-S6KSTDE (UAS-Fur1-sensor/+; Ddc-Gal4/UAS-S6KSTDE)
and (L) UAS-4E-BPAA (UAS-Fur1-sensor/+;
Ddc-Gal4/UAS-4E-BPAA) in DA neurons. GFP corresponding to Fur1
sensor (green) is directly imaged without antibody staining.
(M) Quantification of Fur1 sensor fluorescence intensity relative to TH
for genotypes in (I)-–(L). Each data point represents fluorescence from a
single neuron. n = 50 neurons from 10 PPM1-2 clusters for each genotype. Data
are represented as mean ± SEM. One-way ANOVA with Bonferroni
post-test.
*p < 0.05, **p < 0.01, ***p < 0.001.