Fig 1: MON-2 is required for longevity by regulating trafficking between the Golgi complex and endosomes.(A to C) Microscopic images of transgenic animals expressing mon-2::gfp. The mon-2::gfp transgene was expressed in neurons (arrows) (A), the intestine (B), and neurons and seam cells (an arrow and arrowheads, respectively) (C). odr-1p::rfp was used as a coinjection marker (arrowhead) in (A). Scale bars, 20 µm. (D) Fluorescence microscope images of an intestinal cell in a transgenic animal expressing mon-2::gfp, rfp::rab-10 and the overlay of the two images. Scale bar, 10 µm. (E and F) Colocalization of mammalian GFP-MON2 with giantin, a Golgi marker, was decreased under starvation conditions (E), whereas that with RAB11, a recycling endosome marker, was increased (F) in HeLa cells. Scale bars, 10 µm. (G) Quantification of (E) and (F) (N = 8, 8, 12, and 12, respectively). Error bars represent SEMs (*P < 0.05 and **P < 0.01, two-tailed Student’s t test). Each dot represents a result from a single cell. (H to J) pad-1 RNAi (H), snx-3 RNAi (I), or tbc-1 RNAi (J) reduced the longevity of isp-1(qm150) [isp-1(-)] mutants. See table S5 for specific values and statistical analyses of the life-span data shown in this figure.
Fig 2: MON-2/MON2 appears to increase autophagy by activating LGG-1/GABARAPL2 in C. elegans and mammalian cells.(A to C) mon-2(xh22) [mon-2(-)] consistently decreased the high level of cleaved GFP caused by food deprivation (FD) (A), cco-1 RNAi (B), and isp-1(qm150) [isp-1(-)] mutations (C) (n = 3). Different from consistent results for cleaved GFP, LGG-1–II/I ratio was somewhat variable likely because of subtle separation of LGG-1–I and II bands. Filled arrowhead, LGG-1–I. Open arrowhead, LGG-1–II. Asterisk, cleaved GFP. a-Tubulin, loading control. WT without a transgene, negative control. IB, immunoblot. (D) Small interfering RNA (siRNA) targeting MON2 (siMON2) substantially increased the level of GABARAPL2 while having small effects on those of other tested autophagosomal markers (p62, LC3A, LC3B, LC3C, GABARAP, and GABARAP L1) under basal or starvation conditions (n = 2). One endogenous GABARAPL2 band was detected, which was decreased under starvation and increased by siMON2. In contrast, phosphatidylethanolamine-conjugated (II) and nonconjugated proteins (I) were separated for the other ATG8/LC3 family members. (E) GABARAPL2 was coimmunoprecipitated with MON2 under starvation conditions (n = 3). IP, immunoprecipitation. (F) Immunoblot for GABARAPL2, LC3B, and vinculin in HeLa cells after starvation or starvation with bafilomycin A1 treatment for 6 hours (n = 2). (G) Illustrated fluorescence changes in mRFP-GFP-GABARAPL2 based on its subcellular location in early or late autophagosomes, or autolysosomes. (H) siMON2 in HeLa cells stably transfected with mRFP-GFP-GABARAPL2 under basal or starvation conditions altered the flux of GABARAPL2. Scale bars, 10 µm. (I) The numbers of RFP+GFP+ puncta and RFP+GFP- puncta were counted for each cell and analyzed for statistical differences (N = 21, 21, 22, and 16). Error bars represent SEMs **P < 0.01, Mann-Whitney test).
Fig 3: Endocytic Wls passes through RE. a, Time-lapse images from a video (Movie 4) of a cell co-expressing TagRFP-MON2 and Wls-EGFP. The arrowheads and arrows indicate Wls migrating with MON2. Scale bar, 5 µm. b, Representative confocal images of HA-Wls, EGFP-SNX3, and TagRFP-MON2 in HEK293 cells. The enlarged images show magnified views of the boxed areas. After a 60-min chase, the localization of surface-labeled HA-Wls was observed. The arrowheads indicate HA-Wls that localized at MON2-positive endosomes, in which SNX3-labeled EEs were absent. Scale bar, 10 µm.
Fig 4: Retrograde transport of Wls to the Golgi is impaired in MON2-KO cells. a, Representative confocal images of HA-Wls, EGFP-SNX3, and TagRFP-RAB4B in HEK293 WT or MON2-KO cells. Enlarged images show magnified views of the boxed areas. Images were taken at the indicated times after chasing of surface-labeled HA-Wls. Scale bars, 10 µm. See Supplemental Figure S6a and b for quantitative analyses. b, Representative confocal images of HA-Wls, EGFP-RAB4B, and Scarlet-Giantin in HEK293 WT or MON2-KO cells. The enlarged images show magnified views of the boxed areas. Images were taken at the indicated times after chasing of surfaced-labeled HA-Wls. Scale bars, 10 µm. See Supplemental Figure S6c and d for quantitative analyses.
Fig 5: Subcellular localization of MON2. a, Representative confocal images of MON2. HEK293 cells were transiently transfected with constructs for expression of fluorescent-tagged proteins. Endogenous Golgin97 and GM130 were immunostained as Golgi markers. Enlarged images show magnified views of the boxed areas. Scale bars, 10 µm. b, Quantitative analysis of protein co-localization. MCCs between two proteins were calculated using the ImageJ plugin JACoP. M1 represents the fraction of the red channel that overlapped with the green channel. M2 represents the fraction of the green channel that overlapped with the red channel. The data represent means±SE of the measurements. The numbers of cells used in the calculations are indicated in the parentheses.
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