Novel ethyl methanesulfonate (EMS)-induced alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.
Bottom Line: Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery.Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi.Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.
Affiliation: Department of Neurology, University of California, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.Show MeSH
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Mentions: As we have previously reported, Lrrk localizes predominantly to the membranes of late endosomes and lysosomes, and to a lesser extent to early endosomes, in vivo (Dodson et al., 2012). We thus first asked whether premature follicle cell death in lrrk NS flies is associated with defects in the late endosomal and lysosomal compartments. Using multiple late endosomal and lysosomal markers, including Rab7 (Fig. 3C vs 3B), Lamp1:GFP (Fig. 3C′ vs 3B′) and the acidophilic dye Lysotracker (Fig. 3E vs 3D), we found that many lrrk NS follicle cells showed markedly enlarged late endosomal/lysosomal structures. There did not seem to be any appreciable alteration in lysosome position in lrrk NS flies: the enlarged lysosomes occurred in both the perinuclear region as well as the cell periphery. Expression of the caspase inhibitor diap1 had no significant effect on lysosome enlargement in lrrk NS flies (supplementary material Fig. S3), suggesting that lysosome enlargement is not a consequence of caspase activation. In contrast to the massively enlarged lysosomal structures observed in lrrk NS flies, lrrke03680 homozygous flies showed a mild increase in the size of the Rab7-positive late endosomal compartment in a subset of cells (supplementary material Fig. S2F) and no discernible change in lysosome morphology by Lysotracker staining (supplementary material Fig. S2I) (Dodson et al., 2012). Interestingly, the massive accumulation of Lamp1:GFP within enlarged lysosomes in lrrk NS flies (Fig. 3C′) suggests that their expansion in size is associated with, and perhaps due to, a defect in the degradative properties of the lysosome, because Lamp1:GFP is normally degraded rapidly upon reaching the lysosome (Pulipparacharuvil et al., 2005).
Affiliation: Department of Neurology, University of California, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.