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, Drosophila Lrrk colocalizes with Rab9 at the late-endosome membrane (Dodson et al., 2012). Interestingly, we found that Lrrk-myc co-immunoprecipitated with Rab9-YFP, but did not show significant binding with either YFP alone or with Rab11-YFP (Fig. 7A), which served as controls. Expression of a constitutively active form of rab9 (rab9CA) in follicle cells resulted in marked suppression of the lysosome-enlargement phenotype observed in lrrk NS cells (Fig. 7D vs 7C; Fig. 7E). Similarly, rab9CA significantly, although incompletely, suppressed premature follicle cell death in lrrk NS cells (Fig. 7F). One mechanism by which lrrk could be required for rab9-dependent processes is through regulation of the membrane recruitment of Rab9; however, we did not detect any significant difference in the subcellular localization of Rab9 between the wild-type and lrrk-NS-mutant backgrounds (supplementary material Fig. S6). Collectively, these data suggest that augmenting Rab9 function can, at least in part, bypass the need for lrrk.
Affiliation: Department of Neurology, University of California, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.