Untreated UVC 4 5 Untreated UVC (c) (d) (e) (f) 30 e r 25 p i fo c ll 20 FP ce :G am15 : -1 e g s10 g l f #o 5 0 Nucleic Acids Research, 2012, Vol. 40, No. 16 7921 * * 3-MA (a) 0.45 24 h (b) 0.45 ito 0.4 a r re0.35 t e irm0.3 e p /a0.25 e r na 0.2 a e M0.15 ito 0.4 a r tre0.35 e irm 0.3 e p /ae0.25 r a n 0.2 a e M0.15 0 0 48 h 1 1 2 3 Form Factor 2 3 Form Factor 4 5 24 h Control UVC Figure 3. UVC exposure induces autophagy but no detectable changes in mitochondrial morphology in adult C. elegans. Mitochondrial morphology was assessed using form factor as a measure of elongation (perfect circles = 1) and mean area/perimeter ratio as a measure of interconnectivity. No detectable differences were observed at (a) 24 h or (b) 48 h following UVC exposure. Each data point represents the mean area/perimeter ratio and form factor of mitochondria within a single muscle cell. Mitochondrial morphology was highly variable within each treatment group. Representative images show a (c) tubular, (d) intermediate and (e) fragmented mitochondrial morphology in muscle cells of untreated controls 48 h after UVC exposure. (f) The number of LGG-1::GFP foci per seam cell increased 24 h following UVC exposure compared with untreated controls (Fisher’s PLSD, P = 0.004). Inhibition of autophagy with 3-MA exposure reduced the formation of LGG-1::GFP foci compared with untreated controls (Fisher’s PLSD, P < 0.0001). An overall effect of treatment was determined by ANOVA (P < 0.0001). Bars ± SEM. was signiﬁcant variability in mitochondrial morphology (Figure 3c–e). To evaluate induction of autophagy following UVC exposure, young adult nematodes that express a GFP-tagged LGG-1 protein (71) were exposed to 50 J/m2, and LGG-1::GFP foci were quantiﬁed in hypodermal seam cells 24 h after the exposure. Lgg-1 is the C. elegans homolog to mammalian MAP-LC3 and is incorporated into autophagosomal membranes (71). Appearance of LGG-1::GFP foci in hypodermal seam cells has been extensively used as an indicator of autophagy in C. elegans (71–75). UVC treatment induced a mild but stat- istically signiﬁcant increase in LGG-1::GFP foci 24 h after exposure (Figure 3f). Interestingly, we observed a much greater number of LGG-1::GFP foci in untreated N2 nematodes than has been previously reported (71–75). To conﬁrm that foci were in fact autophagosomal structures, we inhibited autophagosome formation with 10 mM 3- MA, a widely used class III phosphatidylinsositol-3 kinase inhibitor (76), previously shown to inhibit autophagy in C. elegans at this concentration (77). The number of LGG-1::GFP foci decreased signiﬁcantly with 3-MA treatment, indicating that the foci observed in our untreated and UVC-treated nematodes did represent in- duction of autophagy. Therefore we conclude that UVC exposure induced a mild increase in autophagy without persistent changes in mitochondrial morphology. Whether this increase in autophagy signiﬁes a greater mito- chondrial clearance requires further research. Serial UVC exposure results in accumulated mtDNA damage and dose-dependent larval arrest We next sought to investigate the in vivo importance of persistent mtDNA lesions and removal of those lesions. Caenorhabditis elegans develop through four larval stages before reaching adulthood, and mitochondrial function and mtDNA copy number throughout development have been well-characterized (52). Development from L3 to L4 entails a dramatic 3- to 5-fold increase in mtDNA copy number and a switch from anaerobic respiration to OXPHOS, both of which are associated with somatic and germ cell development (52,57). Several researchers demonstrated that either knockdown of ETC components or chemical inhibition of mtDNA replication or protein translation early in larval development (L1 and L2) results in developmental arrest at or before L3 (51–53). The authors concluded that larval development serves as an indicator of overall mitochondrial function. This conclu- sion was further supported by Addo et al. who utilized L3 larval arrest as a screenable phenotype for genes involved in mtDNA maintenance (50). We utilized this L3 arrest phenotype to address the role of removal of persistent mtDNA damage in recovery of mitochondrial function following UVC exposure.