Research ArticleCELL BIOLOGY

A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

See allHide authors and affiliations

Science Advances  16 Dec 2016:
Vol. 2, no. 12, e1601470
DOI: 10.1126/sciadv.1601470
  • Fig. 1 Rab10 distributes to LD-associated structures upon serum starvation.

    (A) Western blot analysis of a purified LD preparation isolated from HuH-7 hepatoma cells, probed with antibodies targeting a variety of organelle markers, including LAMP1 (lysosome), protein disulfide isomerase (PDI) (ER), PLIN2 (LD), and Rab10. Lanes indicate the whole-cell lysate (WCL), postnuclear supernatant (PNS), and LD fraction (LD). (B) Expression of sfGFP-tagged Rab10 or (C) antibody staining of endogenous Rab10 in oleate-loaded Hep3B cells reveals the presence of prominent Rab10-positive structures (green) in close proximity to ORO-stained LDs (red). (D and E) Fluorescence microscopy images of serum-starved HuH-7 hepatoma cells expressing either the constitutively active sfGFP-Q68L (D) or dominant-negative sfGFP-T23N (E) mutant forms of Rab10. Arrowheads indicate examples of Rab10-positive LD-associated structures. (F) Quantification of the number of Rab10-positive LDs observed in n = 3 independent experiments performed on control or HBSS-starved cells (100 cells per condition). ***P ≤ 0.001. (G to I) Comparison of images of a solitary sfGFP-Rab10–positive LD (from a HuH-7 cell HBSS-starved for 1 hour) observed using conventional wide-field epifluorescence (G) or super-resolution microscopy (H and I). The higher resolution of the latter two images (H and I) reveals a membranous structure (I) (sfGFP signal alone) that appears to extend completely around the LD surface. Scale bars, 10 μm (B and C), 5 μm (D and E), and 1 μm (G to I).

  • Fig. 2 Rab10 function is required for LD catabolism.

    (A) Representative immunoblot from n = 3 independent experiments showing the efficiency (>90%) of Rab10 siRNA knockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B and C) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment with either control nontargeting (NT) siRNA or Rab10-directed siRNA and subsequently starved for an additional 48 hours. LDs are stained with ORO, and nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Total ORO-stained area per cell was quantified in (C) from n = 3 independent experiments (>350 cells measured per experiment). (D) HuH-7 cells were treated with control NT siRNA or Rab10 siRNA for 48 hours before re-expression of GFP-tagged forms of wild-type (WT), active (−Q68L), or inactive (−T23N) forms of Rab10. Cells were then serum-starved for a period of 48 hours to look for rescue of the LD breakdown phenotype (total ORO-stained area quantified in n = 3 independent experiments, 30 cells per repeat). (E) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQ was included in the medium during the starvation period (n = 3 independent experiments, 25 cells measured per repeat). (F to I) LDs accumulate in cells isolated from Rab10 KO mice. Cells were preloaded with 400 μM oleic acid overnight. (F) Representative immunoblot of MEFs isolated from WT or Rab10 KO embryos. (G) Measurement of total triglyceride content in WT or Rab10 KO MEFs. (H) Representative fluorescence images of oleate-loaded WT or Rab10 KO MEFs, stained with ORO and DAPI. (I) Digital quantification of average LD content per cell in WT or Rab10 KO MEFs (n = 3 independent experiments, 300 cells per repeat). (J) Quantification of the total LD area per cell in Rab10 KO MEFs transfected with either GFP vector alone or GFP-Rab10 (n = 3 independent experiments, 400 cells per repeat). Cells were preloaded with 400 μM oleic acid overnight. (K) Quantification of the release of [3H]H2O into the medium as a functional readout of mitochondrial β-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starved conditions. Cells were pulse-labeled with [9,10-3H]oleic acid. Data are represented as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; N.S., not significant. Scale bars, 10 μm.

  • Fig. 3 Rab10 is recruited to the LD during starvation-induced autophagy.

    (A to D) Fluorescence images of HuH-7 hepatoma cells expressing sfGFP-Rab10 under resting (A), HBSS-starved (B), Torin1-treated (C), or 3-MA–treated (D) conditions. LDs are stained with ORO (red). Rab10-positive LDs are indicated by arrowheads, and average numbers of Rab10-positive LDs per cell are quantified in (E) (n = 3 independent experiments, 100 cells counted per condition). (F) Quantification of the effect of 3-MA treatment on resting or serum-starved HuH-7 cells (n = 3 independent cells, 100 cells counted per condition). (G) Results of an anti-HA immunoblot from a GTP-agarose bead pulldown of HA-tagged Rab10Q68L or HA-tagged Rab10T23N. (H and I) Immunoblots of Rab10 showing a differential association with GTP beads in HuH-7 cells subjected under resting versus HBSS starvation conditions for 1 hour (H) or treated with DMSO (dimethyl sulfoxide) or 1 μM Torin1 for 1 hour (I). (J) Quantification of Rab10 protein levels from (H) and (I) (n = 3 independent experiments). Data are represented as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale bars, 1 μm.

  • Fig. 4 Rab10-positive LD-associated structures represent nascent autophagic organelles.

    (A to E) Fluorescence images of Hep3B hepatoma cells comparing the colocalization of autophagic and organelle markers with LD-localized Rab10. Boxed areas represent regions of higher magnification. Cells were transfected for 24 hours with mCherry-Rab10 or sfGFP-Rab10, preloaded with 150 μM oleic acid overnight, serum-starved in HBSS for 1 hour, and stained with the LD dye monodansylpentane (MDH) and an antibody to LC3 (A) or transfected to express the autophagic marker GFP-Atg16L1 (B), Rab11 (C), the ER marker Sec61β (D), or the lysosomal marker LAMP1-mCherry (E). Inset number represents the frequency of LD-associated marker that colocalizes with Rab10 in HBSS-starved cells averaged from three independent experiments. Scale bars, 10 μm. (F) Subcellular density gradient fractionation of oleate-loaded Hep3B cells starved in HBSS for 2 hours, lysed (WCL), and further separated into a PNS, CLF, and HSS (high-speed supernatant). The CLF was subsequently loaded onto an 8 to 27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Rab10, a mitochondrial marker (αCOXIV), an endoplasmic reticulum marker (PDI), and the lysosomal resident protein LAMP1.

  • Fig. 5 EM reveals that Rab10-associated autophagosomes extend membrane to envelop adjacent LDs.

    (A and B) Low-magnification EM of LD-autophagosome interactions in HuH-7 cells that were transfected to express an active Q68L form of GFP-tagged Rab10 before starvation in HBSS for 1 hour, followed by fixation and embedding. Scale bars, 1 μm. (A′ and B′) Higher magnification of boxed regions shows autophagic membrane extensions (arrows) moving outward along the LDs during the envelopment process. (C and D) Corresponding low-magnification fluorescence (C) and electron (D) micrographs of a cell exhibiting several prominent Rab10-positive LDs reveal numerous LDs with and without associated Rab10-positive autophagic structures (scale bars, 2 μm). (1 to 3) Correlative EM images of HuH-7 cells expressing active GFP-Rab10 that were cultured on finder grids and photographed by phase and fluorescence microscopy to first locate and identify Rab10-LD–positive structures before fixation and embedding. Boxes show corresponding higher-magnification EM of these structures, confirming the intimate association of Rab10-positive membranes with the engulfed LDs. APM, autophagic membrane.

  • Fig. 6 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. 7 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.

  • Fig. 8 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. 9 Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs.

    (A and B) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. (C) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. (D) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). (E) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. (F) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. (G) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. (H) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring >50 cells per condition per repeat. ***P ≤ 0.001. Scale bars, 1 μm.

  • Fig. 10 Model for the Rab10-EHBP1-EHD2 complex in mediating the autophagic engulfment of an LD during lipophagy.

    Following Rab7-mediated recruitment of degradative machinery to the LD surface, Rab10 works in a complex together with EHD2 and EHBP1 to promote extension of an LC3-positive autophagic membrane along the circumference of the LD surface.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/12/e1601470/DC1

    fig. S1. Rab10 localizes to the LD surface during serum starvation in various hepatocyte cell models.

    fig. S2. Rab10 knockdown results in LD accumulation in multiple cell types.

    fig. S3. Rab10-driven autophagy of LDs is dependent on upstream autophagic initiation.

    fig. S4. Additional EM of Rab10-associated autophagic-LD interactions.

    fig. S5. Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction.

    fig. S6. EHBP1 is an essential component necessary for LD turnover.

    fig. S7. Atg7 knockdown prevents starvation-induced autophagic organelle-LD association.

    fig. S8. Rab7 and Rab10 have different effector protein binding specificities.

    video S1. EHD2 is recruited to a LD after Rab10.

    video S2. mCherry-Rab10 extends along the surface of a LD.

    video S3. GFP-Rab10 extends along the surface of a LD.

    video S4. Rab10 is recruited to a Rab7-positive LD.

    References (49, 50)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Rab10 localizes to the LD surface during serum starvation in various hepatocyte cell models.
    • fig. S2. Rab10 knockdown results in LD accumulation in multiple cell types.
    • fig. S3. Rab10-driven autophagy of LDs is dependent on upstream autophagic initiation.
    • fig. S4. Additional EM of Rab10-associated autophagic-LD interactions.
    • fig. S5. Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction.
    • fig. S6. EHBP1 is an essential component necessary for LD turnover.
    • fig. S7. Atg7 knockdown prevents starvation-induced autophagic organelle-LD association.
    • fig. S8. Rab7 and Rab10 have different effector protein binding specificities.
    • References (49, 50)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • video S1 (.mp4 format). EHD2 is recruited to a LD after Rab10.
    • video S2 (.mp4 format). mCherry-Rab10 extends along the surface of a LD.
    • video S3 (.mp4 format). GFP-Rab10 extends along the surface of a LD.
    • video S4 (.mp4 format). Rab10 is recruited to a Rab7-positive LD.

    Files in this Data Supplement:

Navigate This Article