Fig 1: The Rab10-EHBP1-EHD2 complex mediates the engulfment of LDs by autophagic organelles.(A) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. (B and C) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour siRNA treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 µM oleic acid overnight. (D and E) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 µM oleic acid overnight. (F) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 µM oleic acid overnight. (G and H) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 µM oleic acid overnight. Data are represented as means ± SD. *P = 0.05; **P = 0.01; ***P = 0.001. Scale bars, 1 µm.
Fig 2: Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction.(A) Immunoblot analysis of Hep3B cells transfected to express HA-EHBP1, lysed, and subjected to a GST-Rab10 pulldown. (B) Immunoblot analysis of a GST pulldown experiment in Hep3B cells coexpressing a GST-tagged form of the Rab10-interacting domain of EHBP1 (residues 600 to 902) and HA-tagged Rab10-WT, Rab10-T23N, or Rab10-Q68L. (C and D) Representative immunoblots from pulldown experiments using the same GST-EHBP1 fragment to examine the effect of EHBP1 interactions with endogenous Rab10 after serum starvation (C) or treatment with Torin1 (D) (n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. (E and F) Representative immunoblots from GST-Rab10 pulldown experiments in Hep3B cells, probing for interactions between Rab10 and EHD2 under serum-starved (E) or Torin1-treated conditions (F) (n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. (G) Representative immunoblot of a GST-Rab10 pulldown of EHD2 in Hep3B cells previously treated with either siNT or siEHBP1 and subjected to HBSS starvation for 1 hour (n = 3 independent experiments), quantified in (H). (I) Fluorescence images of EHBP1-positive LDs in HuH-7 cells expressing HA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostaining with anti-HA antibody (green). Cells were preloaded with 150 µM oleic acid overnight. LDs are stained with ORO (red). (J) Fluorescence image of a HuH-7 cell transfected to express GFP-EHD2 (green) and mCherry-Rab10 (red) before serum starvation in HBSS for 1 hour and fixation. The boxed region shows a higher-magnification image of an example of an MDH-stained LD (blue) that is also positive for GFP-EHD2 and mCherry-Rab10. Scale bars, 10 µm. (K and L) Quantification of the appearance of GFP-Rab10–positive LDs (K) or GFP-EHD2–positive LDs (L) in HuH-7 cells following siRNA-mediated knockdown of EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followed by serum starvation in HBSS for 1 hour. Results are from n = 3 independent experiments each and are represented as mean ± SD. **P = 0.01; ***P = 0.001. A total of 80 cells were quantified per condition. (M) Live-cell confocal fluorescence imaging of a starved Hep3B hepatoma cell coexpressing GFP-EHD2 and mCherry-Rab10, depicting the sequential recruitment of Rab10 and EHD2 to MDH-labeled LDs (blue). Note the presence of the mCherry-Rab10–positive structure at the periphery of the LD (arrowhead) before the recruitment of GFP-EHD2, resulting in the emergence of signal overlap by 35 min. Scale bars, 5 µm.
Fig 3: EHD2 and EHBP1 are involved in Rab10-mediated LD breakdown.(A and B) Representative immunoblots showing the efficiency of a 48-hour EHD2 (A) or EHBP1 (B) siRNA–mediated knockdown in the Hep3B hepatoma cell line. (C) Quantification of the effect of EHD2 or EHBP1 knockdown on LD breakdown in Hep3B cells following 48-hour knockdown and 48-hour serum starvation. Average ORO-stained LD area (in pixels) per cell was calculated from n = 3 independent experiments in 100 cells per condition. Cells were preloaded with 150 µM oleic acid overnight. (D) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for 24 hours and then transfecting them with GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown is represented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 µM oleic acid overnight. (E) Subcellular fractionation of oleate-loaded Hep3B cells starved for 2 hours in HBSS through a 0 to 30% discontinuous iodixanol (OptiPrep) gradient. Fractions were collected from the top of the gradient and blotted for EHD2, the endosomal marker Rab5, or the lysosomal marker LAMP1. (F) Representative fluorescence images of basal or serum-starved Hep3B cells treated with siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for 24 hours to measure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloaded with 150 µM oleic acid overnight. (G) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independent experiments, measuring 22 transfected cells per condition. The data are represented as mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001. Scale bars, 10 µm.
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