a novel rab10-ehbp1-ehd2 complex essential for the autophagic … · xiaodong zhang,3 carol a....

SC I ENCE ADVANCES | R E S EARCH ART I C L E

CELL B IOLOGY

1Biochemistry and Molecular Biology Program, Mayo Graduate School, MayoClinic, 200 First Street SW, Rochester, MN 55905, USA. 2Department of Bio-chemistry and Molecular Biology and the Center for Digestive Diseases, MayoClinic, 200 First Street SW, Rochester, MN 55905, USA. 3Department of Bio-chemistry and Molecular Biology, Mayo Clinic Arizona, 13400 E Shea Boulevard,Scottsdale, AZ 85259, USA. 4Department of Internal Medicine, University of Ne-braska Medical Center, Omaha, NE 68198, USA. 5Charles Perkins Centre, Schoolof Life and Environmental Sciences, University of Sydney, Sydney, New SouthWales, Australia. 6Sydney Medical School, University of Sydney, Sydney, NewSouth Wales, Australia.*Corresponding author. Email: [email protected] (M.A.M.); [email protected] (R.J.S.)

Li et al. Sci. Adv. 2016;2 : e1601470 16 December 2016

2016 © The Authors,

some rights reserved;

exclusive licensee

American Association

for the Advancement

of Science. Distributed

under a Creative

Commons Attribution

NonCommercial

License 4.0 (CC BY-NC).

Dow

nl

A novel Rab10-EHBP1-EHD2 complex essential for theautophagic engulfment of lipid dropletsZhipeng Li,1,2 Ryan J. Schulze,2* Shaun G. Weller,2 Eugene W. Krueger,2 Micah B. Schott,2

Xiaodong Zhang,3 Carol A. Casey,4 Jun Liu,3 Jacqueline Stöckli,5

David E. James,5,6 Mark A. McNiven2*

The autophagic digestion of lipid droplets (LDs) through lipophagy is an essential process by which most cells catab-olize lipids as an energy source. However, the cellularmachinery used for the envelopment of LDs during autophagy ispoorly understood.We report a novel function for a small Rabguanosine triphosphatase (GTPase) in the recruitment ofadaptors required for the engulfment of LDs by the growing autophagosome. In hepatocytes stimulated to undergoautophagy, Rab10 activity is amplified significantly, concomitant with its increased recruitment to nascent autophagicmembranes at the LD surface. Disruption of Rab10 function by small interfering RNA knockdown or expression of aGTPase-defective variant leads to LD accumulation. Finally, Rab10 activation during autophagy is essential for LC3recruitment to the autophagosome and stimulates its increased association with the adaptor protein EHBP1 (EH do-mainbindingprotein 1) and themembrane-deforming adenosine triphosphatase EHD2 (EHdomain containing 2) that,together, are essential in driving the activated “engulfment” of LDs during lipophagy in hepatocytes.

oad
on M
arch 1, 2021http://advances.sciencem

ag.org/ed from

INTRODUCTIONMacroautophagy is a catabolic process that results in the delivery of re-usable cellular material to the lysosome for the purposes of energy gen-eration and survival in response to cellular stress. A central organelletargeted by themacroautophagic process is the lipid droplet (LD), a dy-namic, triglyceride-rich structure surrounded by a phospholipidmono-layer. Bound to the surface are different proteins that mediate theformation, dynamics, and catabolism of this organelle (1, 2). LDs aredegraded by a selective form of macroautophagy known as “lipophagy”(3, 4). During lipophagy, a membranous isolation membrane (phago-phore) envelops an individual LD, sequestering it within a double-membrane structure called the autophagosome (5), which subsequentlyfuses with the lysosome to form an autolysosome. Lysosomal lipasesdeposited into autolysosomes can hydrolyze neutral lipids containedwith-in the captured LDs, releasing free fatty acids that can be used for the gen-eration of adenosine 5′-triphosphate via mitochondrial b-oxidation.Although a significant body of knowledge has been compiled regardingthemolecularmechanismsof nonselectivemacroautophagy, surprisinglylittle is known about how LDs are specifically targeted and degraded bythe autophagic machinery for selective lipophagy.

Several small Rab guanosine triphosphatases (GTPases) that activelyregulate membrane dynamics and trafficking (6) have been observed toassociate tightly with the LD phospholipid monolayer (7, 8), althoughthe function of these regulatory enzymes in the regulation of LD functionis poorly understood. We recently reported that Rab7, a regulator ofendocytic vesicle maturation, also plays an instrumental role in the initialrecruitment of degradative compartments to the surface of the LD in

hepatocytes under nutrient-limiting conditions (9). An additional LD-associated Rab protein, Rab10, has also been shown to participate inthe insulin-stimulated translocation of glucose transporter type 4 (GLUT4)in adipocytes (10), basementmembrane secretion (11), and the formationand maintenance of the endoplasmic reticulum (ER) (12). Here, we re-port that Rab10prominently associateswith nascent LC3-positive phago-phores that actively engulf LDs. Rab10 is potently activated underautophagy-stimulating conditions, such as nutrient deprivation ormechanistic target of rapamycin (mTOR) inhibition. This activity triggersthe assembly of a trimeric protein complex at the autophagosome-LDinterface comprising Rab10, the endocytic adaptor EH domain bindingprotein 1 (EHBP1), and the membrane-remodeling adenosine triphos-phatase (ATPase) EH domain containing 2 (EHD2). The assembly of thiscomplex is distinct from the lipophagic action of Rab7 and subsequent toRab7-LD interaction. Thus, Rab10 mediates the selective targeting ofautophagicmachinery to the LDs through a novel trimeric complex thatforms at the “lipophagic junction” between the autophagic machineryand the LD.

RESULTSRab10 distributes to membranous structures intimatelyassociated with hepatocellular LDsIn an independent screen of biochemically fractionated HuH-7 humanhepatocellular carcinoma cells, we found Rab10 to be a bona fidecomponent of isolated LDs, consistent with previous proteomic reportsdemonstrating LD localization (13, 14) (Fig. 1A). To confirm this local-ization, a superfolder green fluorescent protein (sfGFP)–tagged Rab10construct (sfGFP-Rab10) was expressed in several cell types, includingHuH-7 (Fig. 1B)orHep3Bhumanhepatomacells, clone 9 rat hepatocytes,and primary porcine hepatocytes (fig. S1, A toC). In all cell lines examined,we observed that sfGFP-Rab10 distributed to distinct, well-defined, crescent-shaped structures proximal to Oil Red O (ORO)–stained LDs (Fig. 1Band fig. S1). The LD-associated localization of the fluorescently taggedRab10 was also observed by immunofluorescent detection of the endo-genous protein (Fig. 1C). Although expression of aGFP-tagged constitutivelyactive form of Rab10 (GFP-Rab10Q68L) gave rise to numerous Rab10-decorated LDs, these structures were not observed in cells expressing

1 of 16

http://advances.sciencemag.org/


on March 1, 2021


Dow

nloaded from

the dominant-negative form of Rab10 (GFP-Rab10T23N) (Fig. 1, Dto F), indicating that the activity of this GTPase is required for itsrecruitment to the LD. Compared to conventional wide-field fluores-cence microscopy (Fig. 1G), our observations of Rab10 associationwith LDs by super-resolution microscopy (Fig. 1, H and I) providedgreater clarity of these interactions and revealed undulating mem-branous structures within the LD-proximal Rab10 structures thatappeared to completely surround the LD surface (Fig. 1I).

Rab10 participates in LD breakdown undernutrient-limiting conditionsOn the basis of our observation that Rab10 can accumulate onstructures associated with the LD surface, we next assessed whetherRab10 might play a role in LD metabolism. Culture of HuH-7 cells inHanks’ balanced salt solution (HBSS) resulted in significant increases inthe association of Rab10 with LDs in HuH-7 cells (Fig. 1F). To deter-mine whether this increased association was functionally related to LDturnover, we next depleted HuH-7 cells of Rab10 by treatment withsmall interfering RNA (siRNA) (knockdown efficiency, >90%; Fig.2A). After 48 hours, the concentration of serum in the growth mediumwas reduced (conditions known to result in LD loss), andORO stainingwas used to label LDs for optical measurement of total cellular LD con-tent by confocal microscopy. Whereas a 48-hour starvation periodgreatly reduced the LD content of nontargeting control siRNA(siNT)–treated cells (fig. S2D), LD breakdown was significantly attenu-ated by Rab10 depletion, resulting in a nearly twofold retention of LDcontent per cell compared to a siNT-treated control (Fig. 2, B and C). Asimilar response was also observed in additional cell types (fig. S2). Re-expression of GFP-tagged wild-type Rab10 (GFP-Rab10) or the


constitutively active GFP-Rab10Q68L was able to restore LD turnover;however, re-expression of the dominant-negative GFP-Rab10T23N didnot rescue this phenotype (Fig. 2D). Moreover, starvation-inducedRab10-mediated LD breakdown was inhibited in the presence of thelysosomal inhibitor chloroquine (CQ) (Fig. 2E), indicating that Rab10-mediated degradation of LDs occurs in a lysosome-mediated fashion.

To further evaluate the putative role of Rab10 in LD metabolism,mouse embryonic fibroblasts (MEFs) isolated from wild-type or Rab10knockout (KO) mice (Fig. 2F) were lipid-loaded overnight with 400 mMoleic acid to stimulate LD formation and subsequently serum-starved fora further 24 hours to induce LD breakdown. In support of the siRNAfindings, Rab10 KO cells retained nearly fourfold more total triglyceridecompared to MEFs isolated from wild-type mice (Fig. 2G). Further, LDcontent was significantly higher in Rab10 KO MEFs compared to wild-type MEFs, as determined by quantification of ORO staining (Fig. 2, Hand I). LD breakdown in Rab10 KOMEFs was rescued by re-expressionof exogenous GFP-Rab10 (Fig. 2J). To establish a physiological con-nection between Rab10 and lipid metabolism in the context of serumstarvation–induced LD breakdown, we measured b-oxidation inwild-type or Rab10 KO MEFs, which had been pulse-labeled with[9,10-3H]oleic acid and grown in medium containing reduced serum.After just 6 hours of low-serum starvation, Rab10 KOMEFs exhibited anearly twofold reduction in oxidative lipidmetabolism, as quantified bya reduced release of 3H2O into the medium (Fig. 2K).

Autophagy regulates the accumulation of Rab10-positivestructures at the LD interfaceWe postulated that the LD-proximal Rab10-positivemembranous struc-tures observed in nutrient-deprived hepatocytes (Fig. 1) might represent

A C Rab10sfGFP-Rab10B

0

2

4

6

8

10

12

Rab10 Q68L T23N

Rab

-10

po

siti

ve L

Ds/

cel

l

Rest Starve FD GFP-Rab10Q68L E GFP-Rab10T23N

G

H

I

sfGFP-Rab10

WCL

PNSLD

LAMP1

PDI

PLIN2

Rab10

***

***

Wide-field

Super-res

Super-res

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 thewhole-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-loadedHep3B cells reveals the presence of prominent Rab10-positive structures (green) in close proximity toORO-stained LDs (red). (D and E) Fluorescencemicroscopy 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 indicateexamples of Rab10-positive LD-associated structures. (F) Quantification of the number of Rab10-positive LDs observed in n = 3 independent experiments performed on controlor 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 mem-branous structure (I) (sfGFP signal alone) that appears to extend completely around the LD surface. Scale bars, 10 mm (B and C), 5 mm (D and E), and 1 mm (G to I).

2 of 16



on March 1, 2021


Dow

nloaded from

siRNA: NT Rab10

A

D

GADPH

Rab10

Rab10 siRNANT siRNAB

0

2K

4K

6K

8K

10K*

N.S.

GFPGFP

Rab10

Q68L

T23N

Avg

LD

are

a/ c

ell (

px)

siRNA NT Rab10

*

****

**

GFP

GFP

GFP

Rab10

Rab10

siRNA NT Rab10

0

4K

8K

12K

Avg

LD

are

a/ce

ll (p

x)

CQ: – + – – +

0

1

2

3

4

5

6

WT Rab10 KO

T ota

l Tri

gly

ceri

des

(fo

ld c

han

ge)

***

E F

WT MEF Rab10 KO MEFG H

Actin

Rab10

Rab10

KO

WT

0 0.2K0.4K0.6K0.8K1.0K1.2K

WT Rab10 KO

To

tal L

D a

rea/

cel

l (p

x) I **

0 1K2K3K4K5K6K

Tota

l LD

are

a/ce

ll (p

x)

GFP Rab10

*J

0

200

400

600

800

1000

1200

1400

1600

siNT siRab10

Avg

LD

are

a/ce

ll (p

x) **

C

1 0.088

(d

pm

/mg

pro

tein

)

K

Med

ium

[ H3

] H

2O

Rest Starve0

2K

4K

6K

8KWTRab10KO

N.S.

**

Fig. 2. Rab10 function is required for LD catabolism. (A) Representative immunoblot from n = 3 independent experiments showing the efficiency (>90%) of Rab10 siRNAknockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B and C) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment witheither 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 cellsmeasured per experiment). (D) HuH-7 cellswere 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.Cellswere then serum-starved for a period of 48 hours to look for rescue of the LDbreakdownphenotype (total ORO-stained area quantified inn=3 independent experiments, 30cells per repeat). (E) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQwas included inthemedium during the starvation period (n = 3 independent experiments, 25 cells measured per repeat). (F to I) LDs accumulate in cells isolated from Rab10 KOmice. Cells werepreloadedwith 400 mMoleic acid overnight. (F) Representative immunoblot of MEFs isolated fromWT or Rab10 KO embryos. (G) Measurement of total triglyceride content inWTor Rab10 KOMEFs. (H) Representative fluorescence images of oleate-loadedWTor Rab10 KOMEFs, stainedwithORO andDAPI. (I) Digital quantification of average LD content percell 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 GFPvector alone or GFP-Rab10 (n = 3 independent experiments, 400 cells per repeat). Cells were preloadedwith 400 mMoleic acid overnight. (K) Quantification of the release of[3H]H2O into the medium as a functional readout of mitochondrial b-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starvedconditions. 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 mm.

Li et al. Sci. Adv. 2016;2 : e1601470 16 December 2016 3 of 16



http://advances.sciencema

Dow

nloaded from

autophagic isolation membranes or phagophores, known to encapsulatecargo targeted for by autophagy. This would suggest that autophagicsignaling might regulate the recruitment of Rab10 to the LD. To testfor a specific role of autophagy in the initial formation of these Rab10-positive structures, we expressed GFP-Rab10 in HuH-7 cells that werethen subjected to HBSS starvation for 1 hour in the presence or absenceof an autophagy inducer, themTOR inhibitor Torin1 (15). Torin1-treatedcells contained significantly greater numbers of Rab10-positive structuresnear the LD (Fig. 3, A and B). In addition, Torin1 treatment resulted in anapproximately twofold increase in the number of Rab10-labeled LDsobserved in resting cells, an effect that was not further increased by star-vation (Fig. 3E). In contrast, treatment with the autophagic inhibitor3-methyladenine (3-MA) completely abrogated the starvation-inducedaccumulation of fluorescently taggedRab10 near the LD (Fig. 3, D andF). To determine whether Rab10 is activated by autophagic stimulation,as suggested by the accumulation of active GFP-Rab10Q68L on LDs(Fig. 1D), we tested the effect of serum removal or Torin1 treatmenton the GTPase activity of Rab10 using guanosine 5′-triphosphate(GTP)–conjugated beads to pull down active small GTPases (16, 17).We first confirmed that the active Q68L form, but not the dominant-negative T23N form of hemagglutinin (HA)–tagged Rab10, could bepulled down by these beads (Fig. 3G), validating the specificity of thisassay. Lysates of HuH-7 cells subjected to HBSS starvation (Fig. 3H) orTorin1 treatment for 1 hour (Fig. 3I) were subsequently harvested forGTP pulldown. Both treatments resulted in an approximately 2.5-foldincrease in the amount of Rab10 pulled down (Fig. 3J). These data indi-cate thatRab10 can be activated under autophagy-stimulating conditions.

LD-associated Rab10 structures are nascentautophagosomal organellesTo better define the nature of the LD-proximal Rab10-positivestructures and evaluate any potential connection to autophagy, we


examined the colocalization of LC3 or autophagy-related protein 16–like1 (Atg16L1)with the LDanddeterminedwhetherRab10 colocalizedwiththesemarkers of autophagic organelles (Fig. 4,A andB). Immunostainingfor LC3 (Fig. 4A) or coexpression of GFP-tagged Atg16L1 (Fig. 4B) re-vealed that LD-localized Rab10-positive structures can be found to local-ize coincidentwith both of thesemarkers. Consistentwith this finding,wedetected a very close colocalization between LD-localized Rab10 andRab11, another small GTPase with known involvement in phagophoreextension andautophagosomalmaturation (Fig. 4C) (18–22). In addition,we found that LD-localized Rab10 can also colocalizewith the ERmarkerSec61b (Fig. 4D). This is not unexpected, because previous data show thatRab10 plays a key role in the regulation of ER morphology (12) and thatthe ERmembrane itselfmay serve as amajor source ofmembrane for theelongating phagophore (23) (Fig. 4D). Further evidence for a role of Rab10in LD autophagy is demonstrated by the fact that a lysosomal marker,lysosomal-associatedmembrane protein 1 (LAMP1), also exhibits closecolocalization with Rab10 near the LD surface (Fig. 4E). In support ofthese morphological observations, subcellular fractionation of Hep3Bhepatoma cells that hadbeen starved inHBSS revealed a significant overlapbetween Rab10 and LAMP1 in OptiPrep gradient fractions (Fig. 4F).Together, these results suggest that Rab10 accumulates proximal tothe LD following autophagic initiation and participates in the lysosome-mediated turnover of LDs. Consistent with such a model, expression ofconstitutively active GFP-Rab10Q68L was able to drive LD breakdownin siNT-treated hepatocytes but not in cells inwhich autophagic initiationhad first been compromised by siRNA knockdown of Atg7 (fig. S3, A toE). The starvation-induced association of Rab10 with LDs is also pre-vented by Atg7 knockdown (fig. S3, F and G).

To provide greater resolution of the interaction between the putativeRab10-positive nascent autophagosomes and LDs, we conducted elec-tronmicroscopy (EM)ofHuH-7hepatomacells expressing constitutivelyactive GFP-Rab10Q68L that had been either incubated in full-serum

on March 1, 2021

g.org/

0 1 2 3 4 5 6 7 8 9

DMSO Torin1

Rest Starvation **

**N.S.E

Ra

b1

0-p

os

itiv

e L

Ds

/ce

ll

0 1 2 3 4 5 6 7 8

Rest

# R

ab10

-pos

itive

LD

s/ce

ll

*****

Starve Starve3-MA

F

Rab10

Rab10

Actin

Rest

Starv

ation

Input

GTPpulldown Rab10

Rab10

Actin

DMSO

Torin

1

Input

GTPpulldown

G H I

Rest Starvation Torin1CBA

GTPpulldown

Input

HA-Rab

10T23

N

HA-Rab

10Q68

L

0 0.5

1

1.5

2

2.5

3

3.5

Rest

Starv

e

DMSO

Torin

1

Fo

ld c

han

ge

* **

HA (Rab10)

HA (Rab10)

3-MAD

J

Fig. 3. Rab10 is recruited to the LDduring starvation-induced autophagy. (A to D) Fluorescence images of HuH-7hepatoma 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 ofRab10-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 orserum-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-taggedRab10Q68L 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 HBSSstarvation conditions for 1 hour (H) or treated with DMSO (dimethyl sulfoxide) or 1 mM Torin1 for 1 hour (I). (J) Quantification of Rab10 protein levels from (H) and (I) (n = 3independent experiments). Data are represented as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale bars, 1 mm.

4 of 16



on March 1, 2021


Dow

nloaded from

medium or starved for 1 hour in HBSS before fixation and processing.Micrographsof these cells revealed exceptionally intimate associationsbe-tween LDs and electron-dense autophagic membranes (representing au-tophagosomes or potentially lysosomes) that appear to be caught in theact of LD engulfment (Fig. 5, A and B, and fig. S4, A to E). Higher mag-nifications show significant deformation of the autophagic membrane asit extends outward around the LD surface (Fig. 5, A′ and B′). To ensurethat these structures represent the sameRab10-positive features identifiedabove, we starved HuH-7 cells expressing constitutively active GFP-Rab10Q68L inHBSS for 1 hour and subjected the cells to correlative lightand electronmicroscopy (CLEM) by first capturing corresponding phaseand fluorescence images before fixation, embedding, and sectioning forEM. From this approach, we observed an exact correlation between theGFP-Rab10–positive structures at the light level with the lipophagicstructures resolved by EM (Fig. 5, C and D).

Activated Rab10 assembles together with membrane-remodeling adaptors at the LD-autophagic interfaceBecause Rab10 appears to be recruited to the LD under serum starva-tion conditions, we tested whether this association is direct or via othermembrane-adaptor proteins. Given that Rab GTPases exert theirfunctions through the binding of effector proteins, we examinedwheth-er known Rab10 effectors modulate the biogenesis of the autophagicstructures observed on LDs. Recently, it was shown that EHBP1functions as a Rab10 effector during the endocytic recycling of theinterleukin-2 receptor a chain (24). In addition, EHBP1 is known to


regulate endocytosis together with the membrane-remodeling ATPaseEHD2 (25). Therefore, we examined whether these proteins interactwith Rab10 to promote autophagic turnover of LDs. GlutathioneS-transferase (GST)pulldowns andWestern blotting ofHep3Bhepatomacells expressingHA-EHBP1 showed thatGST-Rab10 is able to pull downboth HA-EHBP1 and endogenous EHD2 (Fig. 6A). Consistent with aRab GTPase effector role for EHBP1, a GST-tagged subdomain (residues600 to 902) of EHBP1 (24) is able to pull down the active Q68L form ofRab10 with greater affinity than the inactive T23N form (Fig. 6B). Auto-phagic induction by serum removal or Torin1 treatment, which activatesRab10 (Fig. 3,H and I), also increased the associations betweenRab10 andboth EHBP1 (Fig. 6, C andD) and EHD2 (Fig. 6, E and F). The depletionof EHBP1 by siRNA treatment greatly reduced the associations betweenEHD2 and Rab10 (Fig. 6, G andH, and fig. S5), suggesting that EHBP1 isessential for the interaction between theRab10GTPase and themembrane-deforming ATPase. To determine whether EHBP1 and EHD2 colocalizewith Rab10 at the LD surface under autophagy-stimulating conditions,we expressed HA-EHBP1 or GFP-EHD2 in serum-starved HuH-7 cells.Similar toRab10, both proteins distribute to a structure in close proximityto the LD (Fig. 6, I and J). Cells subjected to EHBP1- or EHD2-targetedsiRNA knockdown contain similar numbers of GFP-Rab10–positivestructures near LDs (Fig. 6K), indicating that Rab10 localizes to the LDindependent of either EHBP1orEHD2. In contrast, the depletionofRab10or EHBP1 by siRNA treatment significantly reduces the accumulation ofEHD2nearLDs (Fig. 6L), indicative of a sequential recruitment of the threeproteins to the surface of the LD.

LD

Rab10 Atg16L1

LD

Rab10 LC3

A B

Rab10

LAMP1 Plin2

EW

CLPNS

CLFHSS1 2 3 4 5 6 7 8 9

OptiPrep fraction

LAMP1

Rab10

COXIV

100

20

15

50PDI

Sec61Rab10

LDDLD

Rab11Rab10

C

F

75%

46% 66%

70%

Fig. 4. Rab10-positive LD-associated structures represent nascent autophagic organelles. (A to E) Fluorescence images of Hep3B hepatoma cells comparing the colo-calization of autophagic and organellemarkers with LD-localized Rab10. Boxed areas represent regions of highermagnification. Cells were transfected for 24 hours withmCherry-Rab10 or sfGFP-Rab10, preloadedwith 150 mMoleic acid overnight, serum-starved in HBSS for 1 hour, and stainedwith the LDdyemonodansylpentane (MDH) and an antibody toLC3 (A) or transfected to express the autophagic marker GFP-Atg16L1 (B), Rab11 (C), the ER marker Sec61b (D), or the lysosomal marker LAMP1-mCherry (E). Inset numberrepresents the frequency of LD-associated marker that colocalizes with Rab10 in HBSS-starved cells averaged from three independent experiments. Scale bars, 10 mm. (F) Sub-cellular 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 super-natant). The CLF was subsequently loaded onto an 8 to 27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected fromthe top of the gradient and blotted for Rab10, a mitochondrial marker (aCOXIV), an endoplasmic reticulum marker (PDI), and the lysosomal resident protein LAMP1.

5 of 16



on March 1, 2021


Dow

nloaded from

To confirm that Rab10 associateswith the LDbefore the recruitmentof EHD2, we performed fluorescence live-cell imaging of Rab10 andEHD2 colocalization with LDs in Hep3B cells (video S1). As depictedin time-lapse stills (Fig. 6M), mCherry-Rab10 (red) is first recruited toone pole of the LD surface, followed by the appearance of EHD2(green). This subsequent colocalization between these two proteins in-creases over the next 5 min (total imaging time, 35 min).

The Rab10-EHBP1-EHD2 complex promotes autophagicengulfment of LDsBecause our evidence is consistent with EHD2 and EHBP1 associatingwith Rab10 as a complex at an autophagic interface on the LD surface,


we next tested whether these proteins functionally contributed to theprocess of lipophagy. Hep3B cells were treated with siRNA for 48 hoursto reduce EHD2 or EHBP1 levels [knockdown efficiency, >90%; repre-sentative blots are shown in Fig. 7 (A and B)] and subsequently serum-starved inmediumcontaining0.2% fetal bovine serum(FBS) for 48hours.The depletion of EHBP1 or EHD2 resulted in an approximately two-fold increase in LD area retained per cell compared to control (Fig.7C). Re-expression ofwild-type EHBP1was able to rescue LDbreakdowninhibited by siEHBP1 treatment (fig. S6A). Likewise, re-expression ofwild-type EHD2, but not an ATPase-dead mutant (EHD2-T72A), wasable to restore LD turnover that had been inhibited by siEHD2 treatment(Fig. 7D). In an effort to disrupt complex stability, we also overexpressed a

LDLD

APM

APM

B'

A B A'

LD

APM

LD

LD APM

APM

LD

1 2 3

C D

1 12 2

33

Fig. 5. EM reveals that Rab10-associated autophagosomes extend membrane to envelop adjacent LDs. (A and B) Low-magnification EM of LD-autophagosome inter-actions 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. Scalebars, 1 mm. (A′ and B′) Highermagnification of boxed regions shows autophagicmembrane extensions (arrows) moving outward along the LDs during the envelopment process.(C andD) Corresponding low-magnification fluorescence (C) andelectron (D)micrographsof a cell exhibiting several prominent Rab10-positive LDs reveal numerous LDswith andwithout associated Rab10-positive autophagic structures (scale bars, 2 mm). (1 to 3) Correlative EM images of HuH-7 cells expressing active GFP-Rab10 thatwere cultured on findergrids and photographed by phase and fluorescence microscopy to first locate and identify Rab10-LD–positive structures before fixation and embedding. Boxes showcorresponding higher-magnification EM of these structures, confirming the intimate association of Rab10-positive membranes with the engulfed LDs. APM, autophagic membrane.

6 of 16



on March 1, 2021


Dow

nloaded from

LD

GFP-EHD2mCherry-Rab10

I J

K

HA-EHBP1

N.S.

0 1 2 3 4 5 6 7 8 9

siNT

siEHBP1

siEHD2

# o

f R

ab10

-po

siti

ve L

Ds/

cell

N.S.N.S.

0 1 2 3 4 5 6 7 8

siNT

siEHBP1

siRab

10

# o

f E

HD

2-p

osi

tive

L

Ds/

cell

*****

N.S.

L

M

mC

herr

y-R

ab10

GF

P-E

HD

2M

DH

(LD

)

pulldown

Input

GST-Rab

10

GST

HA-EHBP1

EHD2

HA-EHBP1

EHD2

GST-Rab10

GST

siNT

siEHBP1

pulldown

Input

EHBP1

EHD2

EHD2

GST-Rab10

pulldown

Input

Rest

Starv

e

EHD2

EHD2

GST-Rab10

Actin

A B C

E F G H

D

0

0.2

0.4

0.6

0.8

1

1.2

siNT siEHBP1

Fo

ld c

han

ge

***

DMSO

Torin

1

EHD2

EHD2

GST-Rab10

pulldown

Input

DMSO

Torin

1

Rab10

Rab10

GST-EHBP1(600–902)

pulldown

Input

Rest

Starv

ation

Rab10

GST-EHBP1(600–902)

GST pulldown

InputRab10

1 2.28

1 2.13

1 6.15

1 5.53

0 min 15 min 30 min 35 min

Rab10 (HA)

Rab10 (HA)

GST-EHBP1(600–902)

pulldown

Input

HA-Rab

10

HA-Rab

10T23

N

HA-Rab

10Q68

L

Fig. 6. Serumstarvationpotentiates the formation of a Rab10 complex togetherwith themembrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction.(A) Immunoblot analysis of Hep3B cells transfected to expressHA-EHBP1, lysed, and subjected to aGST-Rab10pulldown. (B) Immunoblot analysis of aGST pulldownexperiment inHep3Bcells 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 andD) Representativeimmunoblots frompulldownexperimentsusing the sameGST-EHBP1 fragment toexamine theeffect of EHBP1 interactionswithendogenousRab10after serumstarvation (C)or treatmentwith 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) Representativeimmunoblots 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 = 3independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. (G) Representative immunoblot of a GST-Rab10pulldown 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 expressingHA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostainingwith anti-HA antibody (green).Cellswere preloadedwith 150 mMoleic acid overnight. LDs are stainedwithORO (red). (J) Fluorescence imageof aHuH-7 cell transfected to expressGFP-EHD2 (green) andmCherry-Rab10(red) before serumstarvation inHBSS for 1hour and fixation. Theboxed region showsahigher-magnification imageof anexampleof anMDH-stained LD (blue) that is alsopositive forGFP-EHD2 and mCherry-Rab10. Scale bars, 10 mm. (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 knockdownof EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followedby serum starvation inHBSS for 1 hour. Results are from n=3 independent experiments eachand are represented asmean ± 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 cellcoexpressing 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–positivestructure 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 mm.




on March 1, 2021


Dow

nloaded from

siRNA: NT EHD2

EHD2

Actin

Rab10

αLAMP1

αEHD2

αRab5

1 2 3 4 5 6 7 8 9 10 11 12 130% 30%

[Iodixanol]

150

75

25

20

A

B

C D

E

0 2K 4K6K 8K

10K

12K14K16K

siRNA:NT EHBP1 EHD2

Avg

LD

are

a/ce

ll (p

x)

***

0

2K

4K

6K

8K

10K

12K

14K

Avg

LD

are

a/ce

ll (p

x)

siRNA:

GFP GFP GFP EHD2 EHD2 T72A

NT EHD2CQ: − + − − + −

**

**

****

siRNA: NT EHBP1 EHBP1

Actin

0 5

10 15 20 25 30 35

siNT

siRab

10

siEHD2

# R

FP

pu

ncta

/cell **

**G

siNT siEHD2siRab10

50

25

75

100

37

siNT siEHD2siRab10

Ser

um-s

tarv

edB

asal

sta

te

F

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-hourknockdown 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. Cellswere preloadedwith 150 mMoleic acid overnight. (D) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for24 hours and then transfecting themwith GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown isrepresented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 mM 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 collectedfrom the top of the gradient andblotted for EHD2, the endosomalmarker Rab5, or the lysosomalmarker LAMP1. (F) Representative fluorescence images of basal or serum-starvedHep3B cells treatedwith siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for24 hours tomeasure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloadedwith 150 mM oleic acid overnight. (G) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independentexperiments, 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 mm.




on March 1, 2021


Dow

nloaded from

truncated formofEHBP1 that retainsRab10-binding capabilities but lacksthe EHD2 binding domain (GFP-EHBP1600–902); expression of the GFP-EHBP1600–902 construct (but not GFP vector alone) also resulted in an in-crease in retained LDs following serum starvation (fig. S6B). In addition,treatment with the lysosomal inhibitor CQ almost completely abolishedthe effects of EHD2 re-expression (Fig. 7D), providing further evidencethat EHD2-mediated LD breakdown occurs through a lysosome-centricdegradative pathway.

To further define the cellular localization of this complex, we lysedand fractionatedHep3B cells through a 0 to 30%discontinuous iodixanolgradient. Antibodies against EHD2 detected an enrichment of this pro-tein in fractions (fractions 5 to 7) that coincidedwith those enriched in thelysosomal marker LAMP1, providing supporting evidence for EHD2association with autophagic compartments (Fig. 7E).

As an additional functional test to define the role of the Rab10 com-plex in lipophagy, we used a dual-fluorescent variant of the LD-specificmarker perilipin 2 (PLIN2) that reports on the acidity of the environmentsurrounding the LD (9). This reporter, mRFP1-EGFP-PLIN2, fluorescesin both the green and red channels; however, localization of the reporterwithin an acidic lysosome results in quenching of the pH-sensitiveenhanced GFP (EGFP) fluorophore and, thus, leaving detectable fluores-cence of PLIN2 in the red channel alone. This reporter was expressed inHep3B cells that were first subjected to either Rab10- or EHD2-specificsiRNA knockdown. Forty-eight hours later, the number of red-onlyPLIN2-positive puncta was quantified; a reduction in these structuresreflects conditions in which lipophagy has been attenuated. According-ly, the depletion of Rab10 or EHD2 significantly reduced the number ofthese puncta by approximately 50%, indicative of less LD-associatedPLIN2 being internalized within an acidic autolysosomal/lysosomalcompartment (Fig. 7, F and G).

The experiments described above suggest that Rab10 acts at the inter-face between the LD and autophagic machinery to promote membraneextension and envelopment during lipophagy. In an attempt to visualizethis process,wemonitoredRab10-LDdynamics by time-lapsemicroscopy(Fig. 8A and videos S2 and S3). Use of either mCherry- or GFP-taggedRab10 revealed that the Rab10-labeledmembranes actively extend aroundtheLDperimeter (Fig. 8A), reminiscent of ourEMimagingof the intimatemembranous extensions associated with the LD surface (Fig. 5, A′ and B′,arrows). To test whether Rab10, EHBP1, and EHD2 are critical for anautophagic engulfment of LDs, we individually (or jointly) knockeddown each component via siRNA in Hep3B cells. We then monitoredassociation of the autophagic markers LC3 or Atg16L1 with LDs in thecells. Critically, as shown in Fig. 8 (B and C), individual or joint knock-downs of these components significantly affected association of knownautophagic markers with the LD under serum starvation conditions.Furthermore, staining of Atg16L1 or LC3 in Rab10KO MEFs revealedsignificantly decreased association with the LD compared to wild-typeMEFs (Fig. 8, D and E). Direct interactions with the lysosome were alsoattenuated through Rab10, EHD2, or Atg7 knockdown, as demon-strated by decreased LAMP1 associations with the LD in both Hep3BandHuH-7 cell lines (Fig. 8F and fig. S7A). Using control or Rab10 KOMEFs exposed to nutrient deprivation, physical associations betweenLDs and the lysosomal compartment were also observed using LAMP1immunostaining, with phenotypes divided into three groups: none (noassociation), attached (LAMP1puncta docking adjacent to the LD), andengulfed (LAMP1 encircling 60 to 100% of the LD surface) (Fig. 8G).We observed that approximately 15% of the LDs in each starved controlcell showed no association with LAMP1-positive puncta. This lack ofinteraction was increased to 50% of the LDs in Rab10 KO cells. In con-


trast, 60% of LDs in control cells were engulfed by LAMP1-positiveorganelles, whereas only 20% of the LDs in Rab10 KOMEFs showeda similar phenotype (Fig. 8H). Together, these data suggest that thefunctional role of the Rab10-EHBP1-EHD2 complex may be to reg-ulate autophagic membrane extension and promote the engulfmentof LDs during lipophagy.

Rab10 acts downstream of Rab7 to facilitate autophagicdegradation of LDsWe have recently reported a new function for Rab7 as a regulator oflysosomal and multivesicular body (MVB) recruitment to the LD sur-face in hepatocytes (9). To test whether Rab10 and Rab7might performsimilar or distinct functions tomediate lipophagy,we first examinedRab7and Rab10 interactions with their known effector proteins. Rab7 wasfound to interact closelywith itswell-characterized effectorRab7-interactinglysosomal protein (RILP), but not with the Rab10 effector protein EHBP1(Fig. 9A and fig. S8, A and B). In addition to an anticipated binding ofEHBP1, Rab10 was also observed to interact with RILP (Fig. 9B and fig.S8, A and B). However, the Rab7-RILP interaction was unaffected bythe presence of Rab10 because the binding efficiency between thesetwo proteins appeared identical in both wild-type and Rab10 KO cells(Fig. 9C). Furthermore, these Rabs preferentially distribute to differentcompartments in the cell because subcellular fractionation experimentsshow peak distributions of Rab7with LAMP1-positive fractions, whereasRab10 distribution is similar to that of LC3, a classical marker of thephagophore and autophagosome (Fig. 9D).

In agreement with these biochemical findings, we were able to usefluorescently taggedRab10 andRab7 fusion proteins to define differencesin LD association within cells undergoing starvation-induced lipophagy.We examinedHuH-7 cells expressingRab7-GFPbefore serum starvationand 2 hours following HBSS serum starvation, both in the context oftreatment with nontargeting or Rab10-targeted siRNA. No differencein Rab7 localization at the LD surface was observed, regardless ofwhether Rab10 had been knocked down with siRNA (fig. S8, C andD). Using live-cell microscopy, we observed instances of Rab7-positivemembranes extending from LDs that appeared to actively recruit in-comingRab10-positivemembranes to the LD surface (Fig. 9E and videoS4). Of those droplets containing either Rab10 or Rab7, nearly half werepositive for both Rab10 and Rab7, indicating significant interplay be-tween the two Rabs during lipophagy (Fig. 9F). Activation of starvation-induced lipophagy inHuH-7 cells subjected to Rab7 knockdown revealeda significant inhibition in the accumulation of Rab10-positive structures atthe LD surface (Fig. 9G); however, knockdown of Rab10 had no effect onthe percentage of Rab7-positive LDs, suggesting that the two Rabs do notprovide overlapping functions and that Rab10 acts downstream of Rab7(Fig. 9H).

DISCUSSIONHere, we have identified a novel protein complex in hepatocytes that isactivated by nutrient deprivation and responsible for the autophagic en-velopment of LDs during lipophagy. The degradation of these energy-rich structures under nutritionally restricted conditions is dependent onthe accumulation of catalytically activeRab10 at the “lipophagic junction”between theLDandnascent autophagicmembrane (Figs. 1 and2).More-over, the pharmacological or starvation-induced initiation of cellularautophagic pathways enhances Rab10 activity (Figs. 3 and 4). This activa-tion leads to the assembly of a bridging complex consisting of Rab10 andtwo binding partners at the LD interface with the autophagic machinery:

9 of 16



on March 1, 2021


Dow

nloaded from

the adaptor protein EHBP1 and the membrane-deforming ATPaseEHD2 (Figs. 5 to 7). Functional assembly of this complex promotes thesubsequent engulfment of LDs by degradative organelles (Figs. 8 and 9)for the purposes of energy production and cell survival.

Rab10 is a key participant in LD autophagyPreviously considered a nonspecific housekeeping process, macro-autophagy is now appreciated to have the ability to selectively target spe-cific organelles and protein complexes under appropriate conditions.The molecular mechanisms underlying these individual types of selec-


tive autophagy, including mitophagy (26), reticulophagy (27), andpexophagy (28), are currently of intense interest. Despite an increasedfocus on understanding the numerous different types of selective auto-phagy, surprisingly little is known about themolecular targets coordinatingthe process of lipophagy (3).

Recent studies have found that Rab GTPases play a role in con-ventional lipolysis; for example, the knockdown of Rab7 reducesb-adrenergic receptor–stimulated lipolysis in adipocytes (29). In addi-tion, overexpression of Rab18 down-regulates PLIN2 and facilitateslipolysis in HepG2 hepatoma cells (30). Moreover, depletion of Rab32

A

0 s 120 s 220 s 380 s 660 s 1000 s

0 s 80 s 220 s 340 s 460 s 780 sm

Che

rry-

Rab

10G

FP

-Rab

10

0

20

40

60

80

0

20

40

60

80

% L

C3-

asso

c L

Ds/

cell

0

20

40

60

80

siNT

siRab

10

siEHBP1

siEHD2

Tri-siR

NAsiN

T

siRab

10

siEHBP1

siEHD2

Tri-siR

NA

* * * ******

***

0102030405060

siNT

siRab

10

siEHD2

% L

AM

P1-

asso

c L

Ds/

cell

F G LD/LAMP1

*****

0 10 20 30 40 50 60 70 80

None Attach Engulf

% o

f p

hen

oty

pe

WT (#8) Rab10 KO (#15)

*****

N.S.

H

Non

eA

ttach

Eng

ulf

B C D E

% A

tg16

L1-a

ssoc

LD

s/ce

ll

% L

C3-

asso

c LD

s/ce

ll

% A

tg16

L1-a

ssoc

LD

s/ce

ll

0

10

20

30

40

50

WT

Rab10

KO WT

Rab10

KO

****

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 earlytime points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducialpoints of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data frommore than 30 individual cells examined by live-cellimaging. (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 theindicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 mM oleic acid overnight. (D and E) Quantification of the percentage of LDs associated with LC3- orAtg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 mM oleic acid overnight. (F) Quantification of thepercentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring20 cells per condition. Cells were preloaded with 150 mMoleic acid overnight. (G andH) LDs visualized fromWT or Rab10 KOMEFs were divided into three groups on the basis oftheir association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group fromWT and Rab10 KO MEFs. The graphs represent observationsfrom n = 3 independent experiments, measuring 20 cells per condition. Cells were preloadedwith 400 mMoleic acid overnight. Data are represented asmeans ± SD. *P ≤ 0.05; **P ≤0.01; ***P ≤ 0.001. Scale bars, 1 mm.

10 of 16



on March 1, 2021


Dow

nloaded from

0 1 2 3 4 5 6 7 8 9

Resting Starved # R

ab10

-po

siti

ve L

Ds/

cell

***

N.S.

25

2520

2050

70

GST pulldown

Input

GSTWT

GST-RILP

Rab7

Rab7

Actin

GST-RILP

GST-EHBP1

GST-RIL

P

GST

25

2520

2050

GST pulldown

Input

Rab7

Rab7

Actin

25

25

20

20

50

70

50

GST pulldown

Input

Rab10

Rab10

Actin

GST-EHBP1

GST-RILP

GST-EHBP1

GST-RIL

P

GST

35' 55' 60' 70' 85'0'

0

10

20

30

40

50

60

Rab10

Rab7

Rab10

/7

Avg

% R

ab-p

osi

tive

LD

s

A

B

C

D

E

F G

LAMP1

Actin

PLIN2

Rab10

Rab7

LC3-ILC3-II

WCLPNSHSSCLF 1 2 3 4 5 6 7 8 9

150100

50

50

25

202520

15

[OptiPrep]8% 27%

Rest Starve0

20

40

60

Rab

7-p

osi

tive

LD

s/ce

ll

siNT siRab10

N.S. N.S.

HsiNT siRab7

KO WT KO

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 theresults 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 subcellulardensity gradient fractions ofHep3B cells following serum starvation inHBSS for 2 hours, followedby flotation of a CLF through an 8 to 27%discontinuousOptiPrepgradient. Boxesindicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). (E) Live-cell confocal fluorescencemicroscopy of a HuH-7 hepatoma cellcotransfected 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-positivemembrane 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 positivefor the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 fromn= 10 cells. (G) Quantification of the average number of Rab10-positive LDs in resting or serum-starvedHuH-7hepatoma cells treatedwithNT siRNAor Rab7 siRNA. (H) Quantification of the average number of Rab7-positive LDs in resting or serum-starvedHuH-7 cells treatedwithNTsiRNA 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 mm.




on March 1, 2021


Dow

nloaded from

was shown to enhance ATGL expression and lipolysis in hepatocytes(31). Roles for Rabs in macroautophagy have been demonstrated inseveral studies. A functional Rab1, for example, is required for the ini-tiation of autophagosomal biogenesis (32). Rab5 (33) and Rab7 (34)appear to regulate autophagosomal formation and promote the fusionof autophagosomes with lysosomes, whereas Rab11 promotes the fusionofmultivesicular bodies with autophagic vacuoles (18).More recently, wedemonstrated a novel role for Rab7 as a key mediator of lysosomal andMVB recruitment to the LD during lipophagy (9). This current studysuggests that, in response to starvation, another Rab family member,Rab10, is activated and is distributed to autophagic membranes in closeproximity to the surface of LDs. These structures are electron-dense andstain positive for LC3 by immunofluorescence, indicative of a functionin the early autophagic pathway.We propose that Rab7 and Rab10 workcollaboratively during lipophagy (Fig. 9), participating sequentially in aprocess involving the recruitment of autophagicmachinery to the LDsur-face (mediatedbyRab7),which subsequently results in aproductive inter-action that promotes extension of the LC3-positive phagophore alongthe circumference of the LD surface (mediated by the novel Rab10-EHBP1-EHD2 complex; Fig. 10). Together, both lipolysis and lipophagylikely contribute to the catabolism of LDs in the hepatocyte. How thesetwo processes coordinate remains unclear and will require furtherexamination.

Although Rab10 localizes to LDs under nutrient-replete growthconditions, the concomitant activation of Rab10 and its increased local-ization at the LD following starvation or treatment with autophagic in-ducers such as Torin1 (Fig. 3) suggest that the pool of Rab10 residing onthe LD surface under resting conditions might be in an inactive state.However, in response to autophagic signaling, it is unclear whether in-active Rab10 is activated on the LD in situ (similar to Rab7) or whethernewly activated Rab10 is instead recruited to the LD.Onewould predictthat, under nutritional stress, it would be beneficial for the cell to initiallyharvest a triglyceride-containing structure like the LDas an energy sourcebefore the degradationof essential organelles, such asmitochondria or theER. Therefore, the activation of specific Rabs could have important LD-targeting functions for the autophagic machinery.

It is important to add that other closely related Rab proteins mayperform similar functions on the LD. For example, Rab8A and Rab10have highly similar protein sequences (76% identity), underscoringtheir shared cellular functions. In adipocytes, both Rab10 (35) and


Rab8A (36) mediate insulin-stimulated GLUT4 trafficking under theregulation of the same GTPase-activating protein (GAP) AS160. Mostrelevant is a recent study suggesting that Rab8A can mediate LD fusionand growth through an interaction with fat-specific protein 27 (Fsp27)(37). The depletion of Rab8A in Fsp27-overexpressing adipocytes re-duces total LD volume. In contrast, our findings suggest that the deple-tion of Rab10 in serum-starved hepatocytes prevents the breakdown ofLDs, resulting in an overall accumulation of LDs (Fig. 2B). It will be veryinteresting to test the function and contribution of Rab10 to lipophagyand lipid homeostasis in hepatocytes versus adipocytes because itappears that there may be very different roles for autophagy in liverand white adipose tissue lipid metabolism (38–42). In addition, it willbe important to test whether Rab10 plays a specific role in lipophagyor whether it is also involved in other forms of selective autophagy, suchas mitophagy.

The ubiquity of Rabs playing roles atmultiple cellular compartmentsis intriguing. Multipurposing of GTPases is quite common: The con-ventional dynamins act at clathrin pits, lamellapodia, focal adhesions,the trans-Golgi, autophagosomes, and the centrosome/midbody. Theguanine nucleotide exchange factors (GEFs) and GAPs for Rabs are ex-ceptionally diverse and provide distinct functional targeting for each Rabprotein (43), whereas the Ras family of small GTPases, for example, wereoriginally thought to act exclusively at the plasma membrane but arenow appreciated to have important functions on a number of intra-cellular membranes (44). The same can now likely be said for the nu-merous Rab proteins that localize to the LD surface.

A Rab10-EHBP1-EHD2 complex is essential for assembly andfunction of the lipophagic machineryUsing biochemical (Fig. 6, C to F), morphological (Fig. 6, I and J), andlive-cell imaging (Fig. 6M) experiments, we have observed that activatedRab10 exhibits an enhanced binding to both an effector protein (EHBP1)and an ATPase (EHD2) under autophagy-stimulating conditions,supporting the extension of an isolation membrane along the surfaceof the LDunder conditions of nutrient deprivation. The component de-pletion studies (Fig. 6, K and I) suggest that Rab10 is essential for theformation of this complex because it resides on the surfaces of both LDsand autophagic organelles. Activation of Rab10 during cell starvationpromotes the recruitment of the EHBP1 adaptor, required for the as-sociation of the EHD2 ATPase, as graphically depicted by live-cell

Rab10

Rab10

Rab10

Rab10

Rab10

Rab

10

Rab10

Rab10

Rab

10

Rab10

Rab

10Rab

10

Rab10

Rab10

01baR

Rab10

Rab10

7baR

Rab

7

7baRR

ab7

EHBP1

EHBP1 EHBP1

EHBP1EHD2

EH

D2

EHD2

EHD2

EH

D2

LD LD LD LD

APM APM APM APM

Fig. 10. Model for the Rab10-EHBP1-EHD2 complex in mediating the autophagic engulfment of an LD during lipophagy. Following Rab7-mediated recruitment ofdegradative machinery to the LD surface, Rab10 works in a complex together with EHD2 and EHBP1 to promote extension of an LC3-positive autophagic membrane alongthe circumference of the LD surface.

12 of 16




Dow

nloaded from

imaging (Fig. 6M). The participation of EHD2 in this complex is par-ticularly intriguing because it sharesmany features with the dynaminsuperfamily, including its capacity to oligomerize along lipid tubules inring-like structures (45) and actively remodel membranes in response tonucleotide hydrolysis. The use of this membrane-deforming enzymeseems particularly relevant to a process that would require substantialmechanochemical work to deform, stretch, and expand an autophagicmembrane along the LD surface, as observed by fluorescence imaging(Figs. 1, G to I, and 8A) and EM (Fig. 5).

It is possible that this Rab-adaptor-ATPase complex could partici-pate in numerous events related to lipophagy, including the targeting,docking, or expansion/engulfment of the autophagic membrane alongthe surface of the spherical LD; alternatively, it could play importantroles in all three events. In an attempt to identify the specific functionof this complex, we have measured the frequency of associations be-tween the LD and autophagic membrane markers LC3 and Atg16L1(Fig. 8, D and E), as well as the lysosomal marker LAMP1 (Fig. 8H)and the LD in Rab10 KOMEFs, and found fewer numbers of extendedautophagic membranes to be associated with the LD. This suggests thatthe Rab-adaptor-ATPase complex is primarily involved in the regula-tion of LD engulfment by the autophagic membrane and that Rab10depletion in the cell affects the “commitment” step in the autophagicengulfment of LDs during lipophagy. Rab10-mediated direct interac-tions with LAMP1 may also reflect additional degradative mechanismsthat are simultaneously occurring, such as microautophagy. Thesepossibilities warrant further exploration.

In summary, these findings describe a novel complex that appears tomediate the interaction and engulfment of LDs by the autophagic ma-chinery in cells exposed to nutrient stress. These observations furthersupport an important role for autophagy in the utilization of storedcellular lipids but do not rule out a functional synergism between theaction of soluble lipases and the lipophagic machinery. Future in vitrobiophysical studies focusing on the mechanisms by which these com-ponents interact to deform the LD monolayer will prove informative.

on March 1, 2021

MATERIALS AND METHODSCell culture and reagentsThe HuH-7 cell line was a gift from the laboratory of G. Gores (MayoClinic, Rochester, MN). The Hep3B2.1-7 (Hep3B) and clone 9 cell lineswere obtained from the American Type Culture Collection (HB-8064and CRL-1439, respectively). Rab10 KOMEFs were isolated as detailedbelow. Primary pig hepatocytes were a gift from R. Hickey (MayoClinic). HuH-7 andHep3B cells weremaintained inminimumessentialmedium containing Earle’s salts and L-glutamine supplemented with1 mM sodium pyruvate, nonessential amino acids, and 0.075% (w/v)sodium bicarbonate (Corning). Rab10 KO cells were maintained inDulbecco’s modified Eagle’s medium (DMEM). Clone 9 cells weremaintained in F-12K medium. Primary pig hepatocytes were main-tained in DMEM. The medium for all cell lines contained 10% FBS,penicillin (100U/ml), and streptomycin (100 mg/ml) (Life Technologies),and cells were grown at 37°C in 5% CO2. Cells were grown on acid-washed coverslips for fluorescence microscopy and in plastic tissueculture dishes for biochemical analyses. Cells were treated with 150 mM(Hep3B and clone 9) or 400 mM (Rab10 KO MEFs) oleic acid (Sigma-Aldrich) overnight before imaging and biochemical analyses.

The rabbit polyclonal Rab10 antibody was from Sigma-Aldrich(R8906). The mouse monoclonal Rab10 (4E2) antibody was fromGeneTex (GTX82800). The antibody against EHD2 (EPR9821) was


from Abcam (ab154784). The antibodies against EHBP1 were fromAbcam (ab97752) and Proteintech Group (17637-1-AP). The mousemonoclonal LAMP1 (H4A3) antibody was from Santa Cruz Bio-technology Inc. (sc-20011), the LC3 and LC3B antibodies were fromNovus Biologicals (NB100-2220 and NB600-1384, respectively), andthe antibody against PLIN2 was from LifeSpan BioSciences Inc. (LS-B3121). The antibodies against PDI andCOXIV (cytochromeCoxidasesubunit IV) were from Cell Signaling Technology (2446 and 4844, re-spectively), and the antibody recognizing actin was from Sigma-Aldrich(A2066). Secondary antibodies used for immunofluorescence wereconjugated to Alexa Fluor 350, 488, or 594 (Invitrogen), and cells weremounted for microscopy in ProLong antifade reagent (Invitrogen).Secondary antibodies for Western blot analysis were conjugated tohorseradish peroxidase (HRP; Invitrogen). HRP-conjugated reagentsfor Western blot analysis were detected using SuperSignal West PicoChemiluminescent Substrate (Thermo Fisher Scientific).

The autophagy inducer Torin1 was fromTocris (4247). Macroauto-phagywas inhibitedby the additionof 3-MA(10mM,Sigma-Aldrich), andlysosomal proteolysis was inhibited by the addition of CQ (25 mM) (46).

Constructs, siRNA, and transfectionsfGFP was a gift from the laboratory of E. L. Snapp (Albert EinsteinCollege of Medicine). The gene encoding full-length wild-type rat Rab10was inserted into pEGFP-C1, sfGFP-C1, and pmCherry-C3 (a gift fromD. J. Katzmann,MayoClinic). Cloning vector pCMV-HAwas obtainedfromClontech, and pEGX-4T-1was obtained fromGEHealthcare. Theactive human Q68L or dominant-negative human T23N forms ofRab10were cloned into pEGFP-C1 and pCMV-HA. EHD2 andEHBP1were cloned from a HuH-7 complementary DNA (cDNA) library andinserted into pEGFP-C1 and pEGX-4T-1. The constructs encodingLAMP1, LC3, and Atg16L1 were obtained through amplification froma rat brain cDNA library or HuH-7 cDNA library and cloned into thepmCherry-N1 (Takara Bio Inc.) or pEGFP-C1 vectors. LipofectaminePlus reagent (Invitrogen) was used for transfection, according to themanufacturer’s instructions. The siRNA oligos targeting Rab10, EHBP1,EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific,and cells were transfected with the RNAiMAX reagent (Invitrogen). Forre-expression studies, cellswere first treatedwith siRNAoligos for 24hoursand then subsequently transfected with the proper expression constructs.

Generation of Rab10−/− cell linesRab10+/−mice were generated using a Rab10Genetrap embryonic stem(ES) cell line from BayGenomics (#XC820). The gene trap vectorinsertion resulted in a truncated Rab10mRNA comprising amino acids1 of 40 of Rab10 fused to b-galactosidase and neomycin transferase(b-geo). Heterozygous mice were generated by the Australian Phe-nomics Network ES toMouse service at Monash University. Mice weregenotyped by polymerase chain reaction (PCR) and real-time PCRusing the following primers within the gene trap vector: gcggcaccgcgcct-ttcggcgg and ggaagggctggtcttcatccac. Rab10+/− mice were used for timedmatings, and embryos were isolated on embryonic day 14.5.Mouse embry-onic fibroblasts were generated from Rab10+/+ and Rab10−/− embryos andimmortalized by serial passaging, as described previously (47).

Fluorescence microscopyCells were fixed and processed for immunofluorescence using standardprocedures, as described previously (48). Briefly, cells were rinsed inphosphate-buffered saline (PBS), fixed in 3% formaldehyde, and permea-bilized in 0.1%TritonX-100 for 2min.Coverslipswere rinsed thoroughly

13 of 16



on March 1, 2021


Dow

nloaded from

with PBS between treatments. Fixed samples were blocked in buffercontaining 5% goat serum and serially incubated in the appropriateprimary and secondary antibodies at 37°C in blocking buffer. Imageswere acquired using epifluorescence microscopes (Axio Observer andAxiovert 200; Carl ZeissMicroImaging) fittedwith a 63×Carl Zeiss PlanApochromat oil objective [numerical aperture (NA), 1.4] at room tem-perature. Digital images were captured with cameras (Orca II-ERG andOrca II; Hamamatsu Photonics) using iVision software (BioVisionTechnologies). Total LDareawasmeasured using the autosegmentationtool within the iVision software. The numbers of Rab10/EHD2-positivecrescents or red-only PLIN2-positive puncta were both countedmanual-ly. For confocal microscopy, images were acquired with a Zeiss LSM 780confocal microscope (Carl Zeiss MicroImaging). The objective used was a63×/1.2-NA water immersion lens. For super-resolution microscopy,images were acquired bymeans of a Zeiss ELYRA Superresolutionmicro-scopeusing structured illumination.All images and figureswere assembledusing Adobe Photoshop and Illustrator CC software (Adobe Systems).

Starvation conditionsCells were starved in HBSS containing Ca2+/Mg2+ for 1 hour to induceautophagy for quantification of Rab10/Rab7/EHD2-LD association,Rab10-LC3/Atg16/LAMP1/Sec61b colocalization, LC3/Atg16/LAMP1-LD association, andGTP-agarose bead binding assays. Cells were starvedinHBSS for 2 hours for all subcellular density gradient experiments. Cellswere starved in low-serummedium containing 0.2%FBS for 48 hours forLD content quantification.

Time-lapse microscopyHep3B or HuH-7 hepatocytes transfected with GFP-Rab10, as de-scribed above, were plated on glass-bottomed imaging dishes (MatTekCorporation) and starved for 1 hour in HBSS + Ca2+/Mg2+. The cellswere then immediately imaged using a Zeiss LSM 780 confocal micro-scope (Carl Zeiss MicroImaging) at 37°C and 5% CO2. Images wereacquired every 20 s. Movies were converted from 320-pixel × 240-pixelTIF image sequences in Adobe Photoshop CC using H.264 encoding.

Lysosomal enrichmentLysosomeswere enriched using a previously describedmethod (4); briefly,Hep3Bhepatocyteswere grown to a confluence of 90% in 150-mmdishes.Cells were then washed in Dulbecco’s PBS, resuspended in lysis buffer[20 mM tris-HCl (pH 7.4), 1 mM EDTA, 10 mM NaF, and proteaseinhibitors], and homogenized by 20 strokes in a Dounce homogenizeron ice. The lysate was centrifuged at 1000g for 10 min to pellet nucleiand unbroken cells. The postnuclear supernatantwas then collected andcentrifuged at 20,000g to generate a pelleted crude lysosomal fraction(CLF). The CLF was adjusted to 19% iodixanol and layered within an8 to 27% iodixanol gradient. The gradient was then centrifuged at150,000g in a rotor (SW55Ti; Beckman Coulter) for 4 hours at 4°C.Fractions (400 ml) were collected from the top of the gradient. Aliquotswere resuspended in 2× SDS sample loading buffer for loading onto a10% polyacrylamide gel in preparation for Western blotting.

GST pulldown binding assayCells were lysed in TEN100 lysis buffer [20 mM tris (pH 7.4), 0.1 mMEDTA, 100mMNaCl, and cOmplete protease inhibitors (Roche)]. Thepurified GST-fused proteins and purified GST-free proteins fromEscherichia coli were then incubated in cell lysate for 2 hours at 4°C.After washing the beads four times with NTEN300 buffer [20 mM tris(pH 7.4), 0.1 mM EDTA, 300 mMNaCl, 0.05% NP-40, and cOmplete


protease inhibitors (Roche)], samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE). Desired proteins weredetected by immunoblotting.

GTP binding assayCells subjected to starvation or Torin1 treatment were lysed in bindingbuffer [1 mM dithiothreitol, 20 mM Hepes (pH 8.0), 150 mMNaCl,10 mM MgCl2, and cOmplete protease inhibitors (Roche)]. GTP-agarose beads (Sigma-Aldrich) were prewashed three times withbinding buffer and then incubated in cell lysate for 2 hours at 4°C,followed by a further two quick washes with binding buffer. AfterSDS-PAGE and immunoblotting, Rab10 was detected with an anti-body against Rab10 (Sigma-Aldrich).

Triglyceride measurement assayLipid-loaded (400 mM oleic acid) wild-type and Rab10 KOMEFs werestarved in DMEM + 0.2% FBS for 3 hours and homogenized in 100 ml1× PBS containing 0.5% Tween 20. Cell lysates were then incubated at70°C for 5min, followed by spinning at 5000 rpm for 1min at 4°C. Thesupernatant was transferred to a new tube and spun at 14,000 rpm for5min at 4°C. Triglycerides were determined with Infinity TriglycerideReagent (Thermo Fisher Scientific), and proteins were measured withPierce Protein BCA assay (Thermo Fisher Scientific). The normalizationof triglyceridemeasurementwas carried out by dividing triglyceride levelsby protein levels.

b-Oxidation assayFatty acid oxidation was assessed on the basis of 3H2O production from[9,10-3H]oleate. Cells were seeded into six-well plates. One day later,cells were incubated with 2ml of DMEM/10% FBSmedium containingBSA-complexedoleate {0.3mMunlabeledplus [9,10-3H]oleate (1mCi/ml)}overnight. Cells were then washed twice with PBS and incubated with2.5ml of DMEM/10% FBS or DMEM/0.2% FBSmedium. After 6 hours,0.5 ml of the medium was collected for measuring 3H2O production.

Statistical analysis and replication of experimentsNo statistical method was used to predetermine sample size; however,sample sizes used were based on similar experiments from previouslypublished references. The experiments were not randomized, and theinvestigators were not blinded to the experimental allocations or assess-ments of experimental outcomes. Statistical comparisons between twoconditions were assessed by using the two-tailed unpaired Student’st test, where the numerical P values are reported as mean ± SD andrepresent data from a minimum of three independent experiments.The representative images of cells andWestern blots in Figs. 1 (D and E),2 (A, B, F, andH), 3 (A toD andG to I), 6 (C toG), 7 (A, B, and F), and 9(A to C) are examples of multiple similar fields and blots obtained inexperiments that were replicated at least three times (the number of re-plicates are provided in the figure legends).

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/12/e1601470/DC1fig. S1. Rab10 localizes to the LD surface during serum starvation in various hepatocyte cellmodels.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 themembrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction.

14 of 16

http://advances.sciencemag.org/cgi/content/full/2/12/e1601470/DC1

http://advances.sciencemag.org/cgi/content/full/2/12/e1601470/DC1



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)

on March 1, 2021


Dow

nloaded from

REFERENCES AND NOTES1. Y. Guo, K. R. Cordes, R. V. Farese Jr., T. C. Walther, Lipid droplets at a glance. J. Cell Sci. 122,

749–752 (2009).2. T. C. Walther, R. V. Farese Jr., Lipid droplets and cellular lipid metabolism. Annu. Rev.

Biochem. 81, 687–714 (2012).3. R. Singh, S. Kaushik, Y. Wang, Y. Xiang, I. Novak, M. Komatsu, K. Tanaka, A. M. Cuervo,

M. J. Czaja, Autophagy regulates lipid metabolism. Nature 458, 1131–1135 (2009).4. R. J. Schulze, S. G. Weller, B. Schroeder, E. W. Krueger, S. Chi, C. A. Casey, M. A. McNiven,

Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomaltubules in hepatocytes. J. Cell Biol. 203, 315–326 (2013).

5. K. Liu, M. J. Czaja, Regulation of lipid stores and metabolism by lipophagy. Cell DeathDiffer. 20, 3–11 (2013).

6. H. Stenmark, Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10,513–525 (2009).

7. R. Bartz, J. K. Zehmer, M. Zhu, Y. Chen, G. Serrero, Y. Zhao, P. Liu, Dynamic activity oflipid droplets: Protein phosphorylation and GTP-mediated protein translocation.J. Proteome Res. 6, 3256–3265 (2007).

8. P. Liu, R. Bartz, J. K. Zehmer, Y.-s. Ying, M. Zhu, G. Serrero, R. G. W. Anderson, Rab-regulated interaction of early endosomes with lipid droplets. Biochim. Biophys. Acta 1773,784–793 (2007).

9. B. Schroeder, R. J. Schulze, S. G. Weller, A. C. Sletten, C. A. Casey, M. A. McNiven, The smallGTPase Rab7 as a central regulator of hepatocellular lipophagy. Hepatology 61,1896–1907 (2015).

10. H. Sano, G. R. Peck, A. N. Kettenbach, S. A. Gerber, G. E. Lienhard, Insulin-stimulatedGLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotideexchange factor Dennd4C. J. Biol. Chem. 286, 16541–16545 (2011).

11. D. W. Lerner, D. McCoy, A. J. Isabella, A. P. Mahowald, G. F. Gerlach, T. A. Chaudhry,S. Horne-Badovinac, A Rab10-dependent mechanism for polarized basement membranesecretion during organ morphogenesis. Dev. Cell 24, 159–168 (2013).

12. A. R. English, G. K. Voeltz, Rab10 GTPase regulates ER dynamics and morphology. Nat. CellBiol. 15, 169–178 (2013).

13. P. Liu, Y. Ying, Y. Zhao, D. I. Mundy, M. Zhu, R. G. W. Anderson, Chinese hamster ovary K2cell lipid droplets appear to be metabolic organelles involved in membrane traffic.J. Biol. Chem. 279, 3787–3792 (2003).

14. S. Sato, M. Fukasawa, Y. Yamakawa, T. Natsume, T. Suzuki, I. Shoji, H. Aizaki, T. Miyamura,M. Nishijima, Proteomic profiling of lipid droplet proteins in hepatoma cell linesexpressing hepatitis C virus core protein. J. Biochem. 139, 921–930 (2006).

15. C. C. Thoreen, S. A. Kang, J. W. Chang, Q. Liu, J. Zhang, Y. Gao, L. J. Reichling, T. Sim,D. M. Sabatini, N. S. Gray, An ATP-competitive mammalian target of rapamycininhibitor reveals rapamycin-resistant functions of mTORC1. J. Biol. Chem. 284, 8023–8032(2009).

16. D. Bem, S.-I. Yoshimura, R. Nunes-Bastos, F. C. Bond, M. A. Kurian, F. Rahman,M. T. W. Handley, Y. Hadzhiev, I. Masood, A. A. Straatman-Iwanowska, A. R. Cullinane,A. McNeill, S. S. Pasha, G. A. Kirby, K. Foster, Z. Ahmed, J. E. Morton, D. Williams,J. M. Graham, W. B. Dobyns, L. Burglen, J. R. Ainsworth, P. Gissen, F. Müller, E. R. Maher,F. A. Barr, I. A. Aligianis, Loss-of-function mutations in RAB18 cause Warburg Microsyndrome. Am. J. Hum. Genet. 88, 499–507 (2011).

17. R. U. Rehman, E. Stigliano, G. W. Lycett, L. Sticher, F. Sbano, M. Faraco, G. Dalessandro,G.-P. Di Sansebastiano, Tomato Rab11a characterization evidenced a differencebetween SYP121-dependent and SYP122-dependent exocytosis. Plant Cell Physiol. 49,751–766 (2008).

18. C. M. Fader, D. Sánchez, M. Furlán, M. I. Colombo, Induction of autophagy promotesfusion of multivesicular bodies with autophagic vacuoles in K562 cells. Traffic 9, 230–250(2007).

19. H. Knævelsrud, K. Søreng, C. Raiborg, K. Håberg, F. Rasmuson, A. Brech, K. Liestøl,T. E. Rusten, H. Stenmark, T. P. Neufeld, S. R. Carlsson, A. Simonsen, Membraneremodeling by the PX-BAR protein SNX18 promotes autophagosome formation. J. CellBiol. 202, 331–349 (2013).

20. A. Longatti, C. A. Lamb, M. Razi, S.-i. Yoshimura, F. A. Barr, S. A. Tooze, TBC1D14 regulatesautophagosome formation via Rab11- and ULK1-positive recycling endosomes. J. CellBiol. 197, 659–675 (2012).


21. C. Puri, M. Renna, C. F. Bento, K. Moreau, D. C. Rubinsztein, Diverse autophagosomemembrane sources coalesce in recycling endosomes. Cell 154, 1285–1299 (2013).

22. P. Richards, C. Didszun, S. Campesan, A. Simpson, B. Horley, K. W. Young, P. Glynn, K. Cain,C. P. Kyriacou, F. Giorgini, P. Nicotera, Dendritic spine loss and neurodegeneration isrescued by Rab11 in models of Huntington’s disease. Cell Death Differ. 18, 191–200(2010).

23. M. Hayashi-Nishino, N. Fujita, T. Noda, A. Yamaguchi, T. Yoshimori, A. Yamamoto, Asubdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.Nat. Cell Biol. 11, 1433–1437 (2009).

24. A. Shi, C. C.-H. Chen, R. Banerjee, D. Glodowski, A. Audhya, C. Rongo, B. D. Grant,EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans.Mol. Biol. Cell 21, 2930–2943 (2010).

25. A. Guilherme, N. A. Soriano, S. Bose, J. Holik, A. Bose, D. P. Pomerleau, P. Furcinitti,J. Leszyk, S. Corvera, M. P. Czech, EHD2 and the novel EH domain bindingprotein EHBP1 couple endocytosis to the actin cytoskeleton. J. Biol. Chem. 279,10593–10605 (2003).

26. R. J. Youle, D. P. Narendra, Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14(2011).

27. T. Yorimitsu, U. Nair, Z. Yang, D. J. Klionsky, Endoplasmic reticulum stress triggersautophagy. J. Biol. Chem. 281, 30299–30304 (2006).

28. Y. Sakai, M. Oku, I. J. van der Klei, J. A. K. W. Kiel, Pexophagy: Autophagic degradation ofperoxisomes. Biochim. Biophys. Acta 1763, 1767–1775 (2006).

29. A. Lizaso, K.-T. Tan, Y.-H. Lee, b-Adrenergic receptor-stimulated lipolysis requires theRAB7-mediated autolysosomal lipid degradation. Autophagy 9, 1228–1243 (2014).

30. S. Ozeki, J. Cheng, K. Tauchi-Sato, N. Hatano, H. Taniguchi, T. Fujimoto, Rab18localizes to lipid droplets and induces their close apposition to the endoplasmicreticulum-derived membrane. J. Cell Sci. 118, 2601–2611 (2005).

31. Q. Li, J. Wang, Y. Wan, D. Chen, Depletion of Rab32 decreases intracellular lipidaccumulation and induces lipolysis through enhancing ATGL expression in hepatocytes.Biochem. Biophys. Res. Commun. 471, 492–496 (2016).

32. F. C. M. Zoppino, R. D. Militello, I. Slavin, C. Álvarez, M. I. Colombo, Autophagosomeformation depends on the small GTPase Rab1 and functional ER exit sites. Traffic 11,1246–1261 (2010).

33. B. Ravikumar, S. Imarisio, S. Sarkar, C. J. O’Kane, D. C. Rubinsztein, Rab5 modulatesaggregation and toxicity of mutant huntingtin through macroautophagy in cell and flymodels of Huntington disease. J. Cell Sci. 121, 1649–1660 (2008).

34. M. G. Gutierrez, D. B. Munafó, W. Berón, M. I. Colombo, Rab7 is required for thenormal progression of the autophagic pathway in mammalian cells. J. Cell Sci. 117,2687–2697 (2004).

35. H. Sano, L. Eguez, M. N. Teruel, M. Fukuda, T. D. Chuang, J. A. Chavez, G. E. Lienhard,T. E. McGraw, Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulatedtranslocation of GLUT4 to the adipocyte plasma membrane. Cell Metab. 5, 293–303(2007).

36. S. Ishikura, P. J. Bilan, A. Klip, Rabs 8A and 14 are targets of the insulin-regulated Rab-GAPAS160 regulating GLUT4 traffic in muscle cells. Biochem. Biophys. Res. Commun. 353,1074–1079 (2007).

37. L. Wu, D. Xu, L. Zhou, B. Xie, L. Yu, H. Yang, L. Huang, J. Ye, H. Deng, Y. A. Yuan, S. Chen,P. Li, Rab8a-AS160-MSS4 regulatory circuit controls lipid droplet fusion and growth.Dev. Cell 30, 378–393 (2014).

38. C. Wang, Z. Liu, X. Huang, Rab32 is important for autophagy and lipid storage inDrosophila. PLOS ONE 7, e32086 (2012).

39. R. Baerga, Y. Zhang, P.-H. Chen, S. Goldman, S. V. Jin, Targeted deletion of autophagy-related5 (atg5) impairs adipogenesis in a cellular model and in mice. Autophagy 8, 1118–1130(2009).

40. J. Kaur, J. Debnath, Autophagy at the crossroads of catabolism and anabolism. Nat. Rev.Mol. Cell Biol. 16, 461–472 (2015).

41. R. Singh, A. M. Cuervo, Lipophagy: Connecting autophagy and lipid metabolism.Int. J. Cell Biol. 2012, 282041 (2012).

42. Y. Zhang, S. Goldman, R. Baerga, Y. Zhao, M. Komatsu, S. Jin, Adipose-specific deletion ofautophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc. Natl. Acad.Sci. U.S.A. 106, 19860–19865 (2009).

43. J. L. Bos, H. Rehmann, A. Wittinghofer, GEFs and GAPs: Critical elements in the control ofsmall G proteins. Cell 129, 865–877 (2007).

44. J. F. Hancock, Ras proteins: Different signals from different locations. Nat. Rev. Mol. CellBiol. 4, 373–385 (2003).

45. O. Daumke, R. Lundmark, Y. Vallis, S. Martens, P. J. G. Butler, H. T. McMahon, Architecturaland mechanistic insights into an EHD ATPase involved in membrane remodelling.Nature 449, 923–927 (2007).

46. C. de Duve, T. de Barsy, B. Poole, A. Trouet, P. Tulkens, F. Van Hoof, Commentary.Lysosomotropic agents. Biochem. Pharmacol. 23, 2495–2531 (1974).

47. J. Xu, Preparation, culture, and immortalization of mouse embryonic fibroblasts. Curr.Protoc. Mol. Biol. Chapter 28, Unit 28.1 (2005).

15 of 16



48. H. Cao, F. Garcia, M. A. McNiven, Differential distribution of dynamin isoforms inmammalian cells. Mol. Biol. Cell 9, 2595–2609 (1998).

49. R. A. Igal, P. Wang, R. A. Coleman, Triacsin C blocks de novo synthesis of glycerolipids andcholesterol esters but not recycling of fatty acid into phospholipid: Evidence forfunctionally separate pools of acyl-CoA. Biochem. J. 324, 529–534 (1997).

50. J. L. Ramírez-Zacarías, F. Castro-Muñozledo, W. Kuri-Harcuch, Quantitation of adiposeconversion and triglycerides by staining intracytoplasmic lipids with oil red O.Histochemistry 97, 493–497 (2002).

Acknowledgments: We thank E. L. Snapp (Albert Einstein College of Medicine) forproviding the sfGFP vector, M. Czech (University of Massachusetts Medical School) forHA-EHBP1, R. Hickey and S. Nyberg for primary porcine hepatocytes, and members of theMcNiven laboratory, especially G. Razidlo, H. Cao, and L. Qiang, for helpful discussionand input. Funding: This study was supported by grants 5R37DK044650 (M.A.M.),5R01AA020735 (M.A.M. and C.A.C.), and T32DK007352 (R.J.S. and M.B.S.); NIH Challengegrant AA19032 (M.A.M. and C.A.C.); the Robert and Arlene Kogod Center on Aging; andthe Optical Morphology Core of the Mayo Clinic Center for Cell Signaling inGastroenterology (NIDDK P30DK084567). Rab10+/− mouse generation was completed bythe Australian Phenomics Network ES to Mouse service at Monash University and was


supported by National Health and Medical Research Council (NHMRC) project grantsGNT1068469 (J.S.) and GNT1047067 (D.E.J.). D.E.J. is an NHMRC Senior Principal ResearchFellow. Author contributions: Z.L., R.J.S., S.G.W., and M.B.S. performed most of theexperiments, and E.W.K. carried out the EM analysis. X.Z. and J.L. designed and performedthe b-oxidation assay. J.S. and D.E.J. provided Rab10 KO MEF cells. Z.L., R.J.S., S.G.W.,M.B.S., C.A.C., and M.A.M. designed the work, analyzed the data, and wrote the manuscript.Competing interests: The authors declare that they have no competing interests.Data and materials availability: All data needed to evaluate the conclusions in the paperare present in the paper and/or the Supplementary Materials. Additional data relatedto this paper may be requested from the authors.

Submitted 28 June 2016Accepted 15 November 2016Published 16 December 201610.1126/sciadv.1601470

Citation: Z. Li, R. J. Schulze, S. G. Weller, E. W. Krueger, M. B. Schott, X. Zhang, C. A. Casey, J. Liu,J. Stöckli, D. E. James, M. A. McNiven, A novel Rab10-EHBP1-EHD2 complex essential for theautophagic engulfment of lipid droplets. Sci. Adv. 2, e1601470 (2016).

16 of 16

on March 1, 2021


Dow

nloaded from


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

Liu, Jacqueline Stöckli, David E. James and Mark A. McNivenZhipeng Li, Ryan J. Schulze, Shaun G. Weller, Eugene W. Krueger, Micah B. Schott, Xiaodong Zhang, Carol A. Casey, Jun

DOI: 10.1126/sciadv.1601470 (12), e1601470.2Sci Adv

ARTICLE TOOLS http://advances.sciencemag.org/content/2/12/e1601470

MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2016/12/12/2.12.e1601470.DC1

REFERENCES

http://advances.sciencemag.org/content/2/12/e1601470#BIBLThis article cites 50 articles, 14 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science AdvancesYork Avenue NW, Washington, DC 20005. The title (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 NewScience Advances

Copyright © 2016, The Authors

on March 1, 2021


Dow

nloaded from

http://advances.sciencemag.org/content/2/12/e1601470

http://advances.sciencemag.org/content/suppl/2016/12/12/2.12.e1601470.DC1

http://advances.sciencemag.org/content/2/12/e1601470#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


a novel rab10-ehbp1-ehd2 complex essential for the autophagic … · xiaodong zhang,3 carol a....

Documents