Fig. 1. clu overexpression suppresses, and clu loss-of-function exacerbates, PINK1 and parkin null mutant phenotypes in Drosophila.

a–f Confocal microscopy images of the thoracic muscle labeled with mitoGFP (green) and TUNEL (red). a’–f’ Toluidine Blue staining of plastic sections of embedded thoraces. clu overexpression (OE) suppresses muscle death (a–f) and tissue disintegration (a’–f’) in parkin null mutants (park²⁵/dpk²¹), in addition to PINK1 null mutants (PINK1⁵). Expression of UAS-mitoGFP (a–f) and UAS-clu (c, c’, e, e’, f, f’) were driven by Mef2-GAL4. g, h Quantification of muscle death (g) and tissue disintegration (h). For each genotype, 3 male flies were analyzed (mean ± SEM, n = 3). For each fly, 30–50 muscle pieces were analyzed, and the percentage of TUNEL-positive muscle (g) or that of disintegrated muscle (h) was calculated. i–n’ Mitochondria in the muscle are labeled using a mouse anti-ATP5A antibody. Mitochondria are more elongated in clu^d00713 homozygotes (j), as compared to those in wild-type (WT) flies (i). PINK1⁵ (k) and park²⁵/dpk²¹ (l) single mutants show elongated mitochondria with vacuolation. clu^d00713 homozygotes in the PINK1⁵ or park²⁵/dpk²¹ background show exacerbated mitochondrial defects, including enlargement, severe vacuolation, as well as irregular shape and distribution (m–n’). o Quantification of the relative mitochondrial sizes in (i–n’). For each genotype, 3 male flies were analyzed (mean ± SEM, n = 3). For each fly, 30–50 mitochondria were analyzed using Fiji/ImageJ and the average mitochondrial size was calculated. p Results of ATP measurements using whole fly lysates. clu hypo (hypomorph): clu^d00713 homozygotes. Experiments were performed in triplicate (mean ± SEM, n = 3). q A schematic illustration of the genetic interactions between clu and the PINK1–parkin pathway in Drosophila. PINK1 and parkin function in the same pathway to regulate mitochondrial integrity. Loss of either PINK1 or parkin results in severe mitochondrial dysfunction and tissue damage. clu overexpression suppresses phenotypes due to loss of PINK1 or parkin. Partial loss of clu function (clu^d00713 mutants) exacerbates either PINK1 null or parkin null mutant phenotypes. Complete loss of clu function (clu^f04554 mutants) in PINK1 or parkin null mutant background results in lethality. a–f’, i–n’ Scale bars: 5 μM. g–o, p One-way ANOVA with post hoc Tukey’s HSD test.