The arf-like GTPase Arl8 mediates delivery of endocytosed macromolecules to lysosomes in Caenorhabditis elegans

Abstract

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.

Figure 1.

Expression and subcellular localization of fluorescent protein-tagged ARL-8 in worm tissues. (A–C) Expression of ARL-8::GFP driven by the arl-8 promoter in live adult worms. Expression was observed in the pharynx (A), muscle (B), neurons (arrows in A and B), intestine (C), and coelomocytes. A coelomocyte is delineated by a dashed line in C. Bars, 20 μm. (D–G) Analysis of subcellular localization of ARL-8::mCherry in coelomocytes expressing the indicated GFP fusion markers of early endosomes (2xFYVE::GFP; D), early/late endosomes (RME-8::GFP; E), late endosomes/lysosomes (GFP::RAB-7; F), and lysosomes (LMP-1::GFP; G). ARL-8::mCherry was localized to membrane compartments, which were not labeled with 2xFYVE::GFP or RME-8::GFP, but with GFP::RAB-7 and LMP-1::GFP. Some GFP::RAB-7–labeled compartments were negative for ARL-8::mCherry (arrows in F), whereas essentially all LMP-1::GFP–labeled compartments were positive for ARL-8::mCherry. Bars, 5 μm.

Figure 2.

arl-8(tm2504) coelomocytes display supernumerary RAB-7– and LMP-1–positive compartments that are smaller than wild type. (A) Confocal images of arl-8(tm2504)/+ and arl-8(tm2504) coelomocytes expressing the indicated GFP-fusion markers. tm2504 mutation resulted in a decrease in the sizes of the RAB-7– and LMP-1–positive compartments with increase in the number of these compartments. (B and C) Quantification of the number and size of LMP-1–positive compartments in arl-8(tm2504)/+ and arl-8(tm2504) coelomocytes. LMP-1–positive compartments were scored at a single focal plane and sorted into five and three categories according to their number and size, respectively. (D) Rescue of the aberrant morphologies of LMP-1–positive compartments in arl-8 mutants by expression of ARL-8::mCherry under the heat-shock promoters. Adult arl-8 mutants expressing LMP-1::GFP with or without extrachromosomal arrays of phsp::arl-8::mCherry were heat-shocked and observed 24 h after heat shock. Bars, 5 μm.

Figure 3.

sand-1(RNAi) suppresses the formation of supernumerary LMP-1–positive membrane compartments in arl-8 mutants. Nomarski and fluorescence images of LMP-1::GFP–expressing coelomocytes of the arl-8(tm2504)/+, arl-8(tm2504)/+;sand-1(RNAi), arl-8(tm2504), or arl-8(tm2504);sand-1(RNAi). Asterisks show enlarged endosomes. Bars, 5 μm.

Figure 4.

(A) Time-course analysis of endocytic trafficking of TR-BSA in arl-8(tm2504)/+ and arl-8(tm2504) coelomocytes expressing RME-8::GFP. Shown are fluorescence images of the coelomocytes at the indicated time after injection of TR-BSA into the body cavity of the respective worms. (B and C) Endocytosed macromolecules fail to reach ASP-1–enriched compartments in arl-8(tm2504) coelomocytes. Shown are confocal images of the coelomocytes in the respective worms coexpressing LMP-1::GFP and ASP-1::DsRed (top panels in B and C) or confocal images of the coelomocytes 3.5 h after injection of Alexa488-BSA into the body cavity of the respective worms expressing ASP-1::DsRed (bottom panels in B and C). Bars, 5 μm.

Figure 5.

Localization of endocytosed TR-BSA in arl-8(tm2504)/+ (A) or arl-8(tm2504) (B) coelomocytes expressing GFP::RAB-7 or LMP-1::GFP. Shown are confocal images of the coelomocytes 3.5 h after injection of TR-BSA into the body cavity of the respective worms. Bars, 5 μm.

Figure 6.

Time-lapse analysis of endocytic trafficking in coelomocytes. (A and B) Endocytic traffic of Alexa488-BSA to ASP-1–positive compartments in a control (A) or arl-8(tm2504) (B) coelomocyte. Pictures in A and B were extracted from Supplemental Movies 1 and 3, respectively. Bars, 5 μm. (C) Quantitation of fusion events. Average numbers of Alexa488-BSA–containing compartments that fused with a single ASP-1–positive compartment for 30 min were determined in arl-8(tm2504)/+ (n = 12) and arl-8(tm2504) (n = 6) coelomocytes. Error bar, SEM.

Figure 7.

Transient expression of arl-8 promotes the formation of Alexa488-BSA and ASP-1::mCherry double-positive compartments in arl-8 mutants. (A) Alexa488-BSA was injected to ASP-1::mCherry–expressing arl-8 mutants with or without extrachromosomal arrays of phsp::arl-8 transgene and then incubated at 15°C for 15 h before heat shock at 33°C for 30 min. Shown are confocal images of the coelomocytes before heat shock or at the indicated times after heat shock. (B) The formation of Alexa488-BSA and ASP-1::mCherry double-positive compartments was quantified as the percentage of those compartments relative to the total number of Alexa488-BSA–containing compartments in arl-8 mutant coelomocytes (n > 15) with or without extrachromosomal arrays of phsp::arl-8 transgene. Error bars, SEM.

Figure 8.

arl-8 functions upstream to cup-5. (A) Nomarski and fluorescence images of the coelomocyte expressing ASP-1::dsRed or ASP-1::mCherry of the arl-8(tm2504)/+, arl-8(tm2504), cup-5(ar465), or cup-5(ar465);arl-8(tm2504). An asterisk shows an aberrantly enlarged vacuole filled with ASP-1::mCherry in the cup-5(ar465) coelomocytes. Formation of the enlarged vacuoles is strongly suppressed in the cup-5(ar465);arl-8(tm2504) coelomocytes. (B) Quantification of refractile compartments of coelomocytes. Refractile compartments were scored at a single focal plane and sorted into five categories according to their size. Twenty to 25 coelomocytes were scored for each allele. Bars, 5 μm.