galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

ART ICLES

Galectin-3 drives glycosphingolipid-dependentbiogenesis of clathrin-independent carriersRamya Lakshminarayan1,2,3,9,10, Christian Wunder1,2,3,9,10, Ulrike Becken1,2,3,9,10, Mark T. Howes4, Carola Benzing5,Senthil Arumugam1,2,3, Susanne Sales6, Nicholas Ariotti4, Valérie Chambon1,2,3,10, Christophe Lamaze2,3,7,10,Damarys Loew8, Andrej Shevchenko6, Katharina Gaus5, Robert G. Parton4,11 and Ludger Johannes1,2,3,10,11

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How thisprocess is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that acarbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologicallydistinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studiesshowed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins.Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrinwas also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting theexistence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteinswith the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

The endocytic pathway consists of distinct vesicular compartments,which merge in the early endosome. Mechanistically, endocyticevents can be subdivided into clathrin-dependent and clathrin-independent processes1–6. Cargoes that are efficiently internalizedin the absence of clathrin include endogenous surface moleculessuch as glycosylphosphatidylinositol (GPI)-anchored proteins7,CD44 (refs 8,9), major histocompatibility complex (MHC) class Imolecules10, interleukin 2 (IL-2) receptor11, and exogenous ligandssuch as the bacterial Shiga12 and cholera toxins13,14 and simian virus 40(ref. 15). A large fraction of fluid phase is also internalized by clathrin-independent endocytosis (reviewed in refs 2,4). CD44, choleratoxin and fluid-phase markers were mapped to early internalizationstructures with a distinct morphology, termed clathrin-independentcarriers14 (CLICs). CLICs arise directly from the plasma membrane,mature into the GPI-enriched early endosomal compartments7, andsubsequently merge with early endosomes.

Highly organized electron dense coat structures could not bedetected at sites of membrane invagination in clathrin (and caveolin)-independent uptake processes. How cargo proteins are sorted andmembranes are bent to build endocytic pits in these cases has thereforebeen one of the challenging questions in the field of endocytosis.

In mammals, the 15 members of the galectin family of N -glycan-binding proteins have functions in cancer, immunity, inflammationand development16–19. Within this family, galectin-3 (Gal3) is uniquein that it combines a carbohydrate recognition domain in its carboxyterminus with an amino-terminal non-lectin domain that favoursthe formation of Gal3 oligomers including pentamers20,21. BranchedN -acetylglucosamine saccharides that result from the activity of theGolgi-localized β-1,6-N -acetylglycosaminyltransferase V (Mgat5) arepreferred binding determinants for Gal3 in the formation of anextracellular galectin–glycoprotein lattice, which regulates receptortyrosine kinase signalling, cell migration and fibronectin fibrilformation22,23. The availability of Gal3 in tissues is controlled throughexpression and atypical secretion (reviewed in ref. 24). Gal3 expressionis deregulated in human cancers, and it has been suggested that Gal3could be a tumourmarker25,26. Binding toGSLs has been demonstratedfor galectin-4 (Gal4; ref. 27) and galectin-9 (ref. 28), and suggested forGal3 (refs 29,30). The biological functions of these GSL interactionshave remained elusive.

GSLs are present in all mammalian cell types, throughout theanimal kingdom, in bacteria, fungi and plants. Apart from beingcellular receptors for pathogens and pathogenic molecules31 they are

1Institut Curie—Centre de Recherche, Endocytic Trafficking and Therapeutic Delivery group, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 2CNRS UMR3666, 75005Paris, France. 3INSERM U1143, 75005 Paris, France. 4Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia. 5Centrefor Vascular Research, Australian Centre for Nanomedicine and ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NewSouth Wales 2052, Australia. 6Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany. 7Institut Curie—Centre deRecherche, Membrane Dynamics and Mechanics of Intracellular Signaling group, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 8Institut Curie—Centre de Recherche,Proteomics and Mass Spectrometry Laboratory, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 9These authors contributed equally to this work. 10Authors werepreviously members of UMR144 CNRS.11Correspondence should be addressed to R.G.P. or L.J. (e-mail: [email protected] or [email protected])

Received 18 December 2013; accepted 15 April 2014; published online 18 May 2014; DOI: 10.1038/ncb2970

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1

© 2014 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]

mailto:[email protected]

ART ICLES

c Anti-CD44 versus Tf uptake

Control PPMP

Fluo

resc

ence

inte

nsity

(per

cent

age

of c

argo

at

2 m

in)

2 min 10 min

400

300

200

100

∗∗

∗∗

∗∗

∗∗ ∗∗

a

Con

trol

PP

MP

PM

PM

PMPM

PM

g hGal3 clustering

b

Tubules, rings(CLICs)

Vesicles 80–120 nm

HRP-labelled carriers

Car

riers

per

cel

l sec

tion

Control PPMP

∗∗20

15

10

5

Vesicles 40–60 nm

Anti–CD44 uptaked e

Gal3–HRP-labelled carriers

f

Fluo

resc

ence

inte

nsity

(per

cent

age

of M

EB

4)

80

20

40

60

120

100

MEB4

GM95

CG1

ControlPPMP99% CI

r (nm)

800600400200 1,000

60

40

20

80

0

L(r)

– r

–20

Gal3 clustering

Tubules, rings (CLICs)

Vesicles 80–120 nmVesicles 40–60 nm

Control PPMP

∗∗∗∗

60

30

120

90

Max

of L

(r) –

r

0

10

6

2

14

18

Control PPMP

Car

riers

per

cel

l sec

tion

Tf-555CD44Tf-555 CD44

Figure 1 The biogenesis of Gal3-CLICs is GSL dependent. (a) Fluid-phase HRP uptake. MEFs were incubated for 2min at 37 ◦C with HRP(10mgml−1) and processed for electron microscopy. Arrowheads point tovesicular structures of different diameters, and arrows to tubular and ring-shaped CLICs. PM: plasma membrane. (b) Quantification of data as ina on 21–24 cells per condition (n= 3 independent experiments, means± s.e.m.). CLIC numbers were significantly reduced on GSL-depleted(PPMP) cells. (c) CD44 endocytosis in GSL-depleted cells. Quantification ofinternalized anti-CD44 antibodies and transferrin (Tf) on control or PPMP-treated MEFs after incubation for 2 or 10min (15–20 cells per condition,n=3 independent experiments, means ± s.e.m.). CD44 uptake was stronglyinhibited in PPMP-treated cells. (d) Experiment as in c (2min uptake) onGSL-deficient GM95 cells, maternal MEB4 cells, and a rescue clone (CG1).CD44 uptake was strongly inhibited in the absence of GSLs (81–90 cellsassessed from 9 fields per condition, means, n=2 independent experiments).(e) Electron microscope tomography. MEFs were incubated for 2min at37 ◦C with Gal3–HRP, and processed for electron microscope tomographyin the presence of ascorbic acid to quench plasma membrane accessibleHRP. Intracellular Gal3–HRP localized in structures with CLIC morphology.

The boxed region in the left panel signifies the part of the image that isrepresented in the tomographic reconstructions (green). The top middle panelshows a tomographic reconstruction of the CLIC structure (green) superposedonto the image from the left panel. The middle and right columns showtomographic reconstructions of the CLIC structure represented from differentangles. (f) GSL dependency of Gal3-CLIC formation. Experiment as in e oncontrol or PPMP-treated TECs (18–20 cells per condition, n=3 independentexperiments, means ± s.e.m.). Gal3-CLIC (tubules/rings) formation wasstrongly inhibited on PPMP-treated cells. (g,h) Clustering of Gal3–Alexa647in control and PPMP-treated HeLa cells. Mean Ripley’s K-function curves,L(r)− r , derived from single-molecule localizations obtained with dSTORMimaging, plotted against radius, r , of concentric circles centred on eachmolecule relative to random distributions (99% confidence interval (CI)of simulated data) (g), and maxima of Ripley’s K-function curves (h).In g, data are averages of 10–20 cells, n= 3 independent experiments.In h, each symbol represents one image region; small horizontal linesindicate mean (± s.e.m., n = 3 independent experiments). Statisticalanalysis in this figure: Student’s unpaired t-test, ∗∗P<0.01, ∗∗∗∗P<0.0001.Scale bars, 200nm.

critical for cell adhesion, migration and signalling32–34. Identifyingmechanism(s) by which GSLs regulate these cellular functions is oneof the major challenges in membrane biology research.

Here, we found thatGal3 binds to theCLIC cargo proteinCD44 andcontrols its GSL-dependent endocytosis, as well as that of β1-integrin.On the basis of cell and model membrane work, we propose anintegrated model for the function of galectins and GSLs in cargo

recruitment and the construction of endocytic pits in the biogenesisof CLICs.

RESULTSGal3 is localized to CLICsTo analyse the contribution of GSLs to endocytic uptake in mam-malian cells, we incubated mouse embryonic fibroblasts (MEFs) for

2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION


ART ICLES

aGal3–HRP-labelled carriers

Tubules, rings (CLICs)

Vesicles 80–120 nm

Vesicles 40–60 nm

Car

riers

per

cel

l sec

tion

∗∗

20

15

10

5

DMSO Lat ADynol siRNA clathrin

Cav1–/–No. 1 No. 231-2 34-2

b

Gal3 endocytosis

ControlLatrunculin A

10080604020Fluorescence intensity(percentage of control)

∗∗∗

Gal

3P

hallo

idin

Mer

ge

Control Latrunculin A

c

Figure 2 Gal3 uptake shows hallmarks of clathrin-independent endocytosis.(a) Gal3–HRP CLIC formation. MEFs were incubated for 2min at 37 ◦Cwith Gal3–HRP, and processed for electron microscopy (presence ofascorbic acid) in the following conditions: 0.1% dimethylsulphoxide(DMSO), 1 µgml−1 latrunculin A (Lat A), 10 µM dynoles 31-2 or 34-2,or depletion of clathrin heavy chain using siRNAs. Cav1−/− MEFs werefrom caveolin 1 knock out mice. 18–21 cells per condition were analysed.Gal3-CLIC formation was significantly inhibited only in the presence of

latrunculin A (means ± s.e.m., n=3 independent experiments). (b,c) Gal3endocytosis under conditions of perturbed actin polymerization. Gal3was incubated for 15min at 37 ◦C with TECs that were treated (rightpanel) or not (left panel) with 1 µM latrunculin A. Gal3 endocytosiswas significantly inhibited on latrunculin A treatment. 25–30 cells percondition (means ± s.d., n = 3 independent experiments). Statisticalanalysis in this figure: Student’s unpaired t-test, ∗∗P<0.01, ∗∗∗P<0.001.Scale bars, 10 µm.

2minwith a fluid-phasemarker, horseradish peroxidase (HRP). Basedon electron microscopy analysis, three classes of uptake structurescould be distinguished in control cells8: 40–60 nm and 80–120 nmvesicles corresponding to caveolar or clathrin-mediated endocyto-sis, respectively (Fig. 1a, arrowheads) and tubular and ring-shapedCLIC structures (Fig. 1a, arrows). When GSLs were depleted us-ing an inhibitor of glycosylceramide synthase (D,L-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, PPMP), only the occur-rence of tubular and ring-shaped CLIC structures was significantlyreduced (Fig. 1a,b), strongly suggesting that CLIC processes wereexquisitely sensitive to GSL levels. Consistent with this, the uptakeof the CLIC cargo protein CD44 (ref. 8) was almost completely abol-ished in GSL-depleted cells, whereas transferrin (Tf) uptake throughthe clathrin pathway was not affected (Fig. 1c and SupplementaryFig. 1a,e). To rule out potential off-target effects of pharmacologicalGSL depletion, we repeated these experiments onGSL-deficientGM95cells35. CD44 uptake was also strongly diminished on these cells,when compared with parental GSL-containing MEB4 cells (Fig. 1dand Supplementary Fig. 1b,e). Stable overexpression of the ceramideglucosyltransferase-1 gene (CG1 cells) in GM95 cells partially rescuedthe CD44 uptake phenotype. CD44 surface levels were not alteredin these different conditions (Supplementary Fig. 1c,d), most likelybecause CD44 is predominately localized at the plasma membrane atsteady state. Thus, uptake inhibition under conditions of pharmaco-logically or genetically induced GSL deficiency clearly pointed to afunctional role for GSLs in CD44 internalization and CLIC biogenesis.

To establish a molecular link between GSLs and CLICs, wesearched the CLIC proteomics list8 for proteins with a clearlyestablished capacity to bind carbohydrates. Gal3 was one of thebest candidates24. We expressed recombinant Gal3, which for someexperiments was tagged with HRP or fluorophores. In MEFs (Fig. 1eand Supplementary Video 1) or mouse mammary tumour epithelialcells (TECs; Supplementary Video 2), HRP-coupled Gal3 decoratedsmall crescent-shaped cisternae with invaginations, exhibiting thecharacteristic hallmarks of CLICmorphology14. These structures wereformed with as little as 10 ngml−1 of Gal3–HRP (SupplementaryFig. 1f), a concentration that is within the range of Gal3 levels inhuman serum36,37. Depletion of GSLs caused a pronounced loss ofthese Gal3-CLIC structures (Fig. 1f and Supplementary Fig. 1g). Takentogether, the data established a link between GSL expression, Gal3endocytosis and CLIC formation.

To address the contribution of GSLs to Gal3 organization beforeuptake, we determined Gal3 distribution at the plasma membrane bydSTORM super-resolution localization microscopy. PPMP treatmentrevealed that Gal3 clustering at the cell surface was GSL dependent(Fig. 1g,h and Supplementary Fig. 2a–k andNote 1). Gal3 clusters were75 ± 2 nm in diameter, and became smaller and more abundant inGSL-depleted cells.

Apart from being clathrin independent, CLIC processes are alsolittle reliant on the scission GTPase dynamin, but strongly sensitiveto actin perturbation14. We found here that Gal3 uptake was largelyunaffected by inhibition of clathrin and dynamin activities, both



ART ICLES

in TECs and HeLa cells (Supplementary Fig. 3a–d). The genesis ofGal3-CLICs in MEFs was also not altered under these perturbationconditions (Fig. 2a). In contrast, a marked reduction of Gal3uptake (Fig. 2b,c) and Gal3-CLIC formation (Fig. 2a) was observedwhen actin polymerization was inhibited with latrunculin A. Thesefindings further confirmed that Gal3 was internalized in a clathrin-independent manner through CLICs.

In conclusion, Gal3 clustering, CD44 uptake and Gal3-CLICformation required GSL expression.

Gal3 needs glycosylated proteins for binding to cellsGal3 binding to TECs was not affected by GSL depletion(Supplementary Fig. 4a), suggesting that proteins were the primarybinding determinants. Indeed, removal of the extracellular domainsof glycosylated and non-glycosylated plasma membrane proteinswith proteinase K resulted in strongly decreased Gal3 binding,whereas binding of the B-subunit of cholera toxin (CTxB) to theGSL GM1 was not affected (Supplementary Fig. 4b). A similarconclusion was reached when TECs were treated with tunicamycinor 1-deoxymannojirimycin to inhibit protein N -glycosylation.Again, Gal3 binding to the surface of cells was strongly reduced,whereas binding of CTxB to its lipid receptor GM1 was not affected(Supplementary Fig. 4c). Thus,N -glycosylated proteins were requiredfor Gal3 recruitment to the plasma membrane.

Gal3 interacting proteins at the cell surface were identified by Gal3pulldown followed by mass spectrometry. The list of Gal3 binderscontainedmostly proteins from plasmamembrane, extracellular spaceor intracellular compartments (cytosol, lysosomes, Golgi apparatus)that communicate with the plasmamembrane (Supplementary Fig. 4dand Table 1), and included several known Gal3 interactors (Fig. 3a,red bars). Strikingly, putative and established CLIC cargoes, includingCD44, were also found on the list of Gal3 binders (Fig. 3a,asterisks). The interaction between Gal3 and CD44 was confirmedby western blotting (Fig. 3b). Furthermore, we showed that 79%(means, n= 2 independent experiments, with 16–18 cells assessedfrom 6 fields) of antibody-labelled endogenous Gal3 co-localizedwith endocytic structures that were positive for endogenous CD44(Fig. 3c). Likewise, exogenous Gal3 co-localized with endogenousCD44 at early times of uptake (Fig. 4a,b). Both experiments stronglysuggested that Gal3 and CD44 were internalized by the sameendocytic carriers.

Gal3 is required for CLIC formationTo test the functional implication of Gal3 in CLIC formation, thelectin was depleted from TECs using distinct short interfering RNAs(siRNAs; Supplementary Fig. 5a,d). Although CD44 cell surface levelswere not affected in Gal3-depleted cells (Supplementary Fig. 5b),CD44 uptake was strongly inhibited (Fig. 4c,d and SupplementaryFig. 5c). Addition of exogenous Gal3 rescued CD44 uptake (Fig. 4c) ina dose-dependent manner (Fig. 4d). Significant rescue was obtainedwith the serum-level Gal3 concentration of 10 ngml−1, and maximalrescue at 1 µgml−1, which is at the lower end of Gal3 binding affinitiesfor carbohydrates with optimal branching29, or for serum proteins38.Back-addition of 0.8 µgml−1 of exogenous Gal3 to Gal3-depleted cellsyielded endogenous labelling intensities (Fig. 4e,f), indicating that thisconcentration indeed restored natural Gal3 levels in cells. When Gal3

was added back at 10 µgml−1 to Gal3-depleted MEFs, CD44 uptakewas not rescued any more (Fig. 4d). It is likely that at such highconcentrations, glycoproteinswere crosslinked into an extended latticethereby preventing uptake.

AnN-terminal deletionmutant of Gal3 that lacks critical clusteringdeterminants (termed Gal31Nter; ref. 39) was much less efficientat rescuing CD44 uptake (Fig. 4d), strongly suggesting that Gal3oligomerization was of critical importance for its endocytic function.Interestingly, Gal3 failed to rescue CD44 uptake on GSL-depleted cells(Fig. 4c). These findings established a functional link between Gal3and GSLs in CD44 endocytosis.β1-integrin is one of the most predominant interacting partners

of Gal3 (ref. 40; Fig. 3a,b), and the protein was identified as a tophit of CLIC-associated proteins by mass spectrometry8. Here wefound that its endocytic uptake was inhibited in Gal3-depleted MEFs(Fig. 4g and Supplementary Fig. 5d,e). The uptake of β1-integrinwas also sensitive to clathrin depletion (Fig. 4g and SupplementaryFig. 5d,e), suggesting the existence of multiple uptake routes thatmay be selected on the basis of extra- and intracellular conditions.β1-integrin surface levels were similar in all conditions, except for aslight reduction in the clathrin and Gal3 double depletion condition(Supplementary Fig. 5f).

Depletion of Gal3 also reduced the formation of fluid-phaseHRP-labelled CLICs (Fig. 4h and Supplementary Fig. 5g). As theimpairment was only partial, we concluded that many but notall CLICs required Gal3 for their biogenesis. Similar conclusionswere reached when lactose was used as a competitive inhibitor ofgalectin function: the inhibitory effect on CD44 uptake was verystrong (Supplementary Fig. 6a–c), whereas fluid-phase HRP CLICformation (Supplementary Fig. 6d,e) and dextran fluid-phase uptake(Supplementary Fig. 6f,g) were only partly reduced.

In summary, Gal3 was recruited to N -glycosylated proteins, wasrequired for uptake of CD44 and β1-integrin, and for CLIC formation.

N-glycosylation of CD44 required for uptakeCD44 is a glycoprotein with 5 N - and at least 7 O-glycosylationsites41,42. Inhibition of N -glycosylation using 1-deoxymannojirimycinreduced the average density of CD44 molecules in clusters, and CD44cluster size (Fig. 5a,b and Supplementary Fig.7a,b). CD44 uptakewas strongly perturbed in MEFs treated with the N -glycosylationinhibitors 1-deoxymannojirimycin, kifunensin or swainsonine(Fig. 5c and Supplementary Fig. 7c; see Supplementary Fig. 7d,e forunchanged CD44 surface levels). In addition, we generated a CD44mutant for which all 5 N -glycosylation sites were eliminated. ThisCD44-5xN-Glyc mutant failed to be taken up into cells (Fig. 5d,e; seeSupplementary Fig. 7f,g for unchanged CD44 surface levels). Thesefindings clearly established the importance of N -glycosylation onCD44 clustering and uptake.

Gal4 also localizes to CLICsGal4 binds to GSLs (ref. 27) and forms cluster at the plasmamembrane43. We therefore investigated whether Gal4 could alsobe linked to the CLIC pathway. A proteomics analysis of Gal4interacting partners yielded a markedly different profile comparedwith that of Gal3 (Fig. 3a, bottom part, and Supplementary Fig. 4dand Table 1). This reflects the distinct preference of Gal3 and Gal4



ART ICLES

a

β1-integrin

CD44

IFNAR2

Actin

10 5 2 –Gal3 +Gal3

Lysate (μg) Eluateb c

Overlay

Zoo

m

Anti-CD44Anti-Gal3

40

0

10

30

20

β1 -integrin

CD44

Known interaction partners

CLIC proteins

Num

ber

of p

eptid

esN

umb

er o

f pep

tides

1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Gal4 interaction partners

α5 -integrin

Gal3 interaction partners

0

10

30

20

50

∗

∗

∗∗∗ ∗

∗ ∗ ∗∗

∗

∗

Figure 3 Gal3 and Gal4 interacting partners. (a) Proteomics analysis of TECproteins that were pulled down with Gal3 or Gal4. Hits were classifiedaccording to the number of peptides per protein. Red columns: knownbinding partners; asterisks: proteins from the CLIC proteome8. The numberingon the x axis relates to Supplementary Table 1. The interaction profilesfor each galectin were specific, with some overlap. (b) Western blottinganalysis for Gal3-binding proteins. Clear interactions could be detected

for β1-integrin and CD44. Negative controls: interferon α receptor chain 2(IFNAR2) and actin. Representative results, n=3 independent experiments.(c) Endocytic uptake assay (10min) of antibodies against endogenous Gal3and endogenous CD44 in TECs. 79% (mean, n=2 independent experiments,16–18 cells each) of anti-CD44-positive structures also contained anti-Gal3-specific immunolabelling. Scale bars in c: top, 10 µm; bottom, 2 µm.Uncropped images of blots/gels are shown in Supplementary Fig. 8.

for complex carbohydrates whereby the β-galactosidic linkage in[-3Galβ1-4GlcNAβ1-]n repeats is specific for Gal3 (ref. 29), andsulphated galactosylceramides for Gal4 (ref. 44). At early times ofuptake (30 s), most intracellular carriers indeed contained eitherGal3 (green) or Gal4 (red; Fig. 5f). Quantification using Manders’coefficients revealed that only 35.1 ± 3.6% of Gal3 structures werealso Gal4 positive, and 56.7% ± 11% of Gal4 structures were alsoGal3 positive. In contrast, two differentially labelled Gal3 moleculesco-localized extensively (Fig. 5g), and co-distribution was calculatedto be 84.1% ± 1.1% (means ± s.d., n= 3 independent experiments

24–30 cells each). Gal3 and Gal4 thus associated with overlapping butdistinct endocytic environments.

On the basis of electron microscopy analysis, HRP-conjugatedGal4 strongly localized to very early endocytic carriers with tubularand ring-shaped CLIC morphologies (Fig. 5h). Its distributionbetween CLICs and other uptake structures was similar to thatof Gal3 (compare Figs 1f and 5i). In combination with theimmunofluorescence data on limited overlap of Gal3 and Gal4 at earlytimes of uptake (Fig. 5f), we concluded that Gal3 and Gal4 most likelylabelled different CLIC populations.



ART ICLES

g

Fluo

resc

ence

inte

nsity

(per

cent

age

of s

cram

ble

d)

40

80

60

20

100

Scrambled

Anti-β1-integrin uptake h

Car

riers

per

cel

l sec

tion

20

15

10

5

Scrambled Gal3siRNA1

Gal3siRNA2

–

Tubules, rings(CLICs)Vesicles 80–120 nmVesicles 40–60 nm

HRP-labelled carriers

Fluo

resc

ence

inte

nsity

(per

cent

age

of M

EF

WT

)

40

80

60

20

100

0 0.8

MEF Gal3–/–

120

140

e

0.2

Gal3 labelling

MEF Gal3–/–

+0 μg ml–1 Gal3

MEF WT

Endogenous Gal3

MEF Gal3–/–

+0.8 μg ml–1 Gal3

f

Control

d

Fluo

resc

ence

inte

nsity

(per

cent

age

of c

ontr

ol)

80

20

40

60

Gal3 WTGal3ΔNter

120

100

∗∗

∗∗

∗∗∗∗

∗∗∗∗∗

∗

∗∗

∗∗

Anti-CD44 uptake

Gal3 siRNA3Gal3 (μg ml–1)

+ + + + +– – 0.01 0.1 1 10

Gal3 co-labelling

Anti-CD44

Percentage of co-localization

2

8070605040302010

Tf–Alexa488

10

40Tim

e (m

in)

b

+

+

+

+

+

Gal

3

––

––

–

c

Fluorescence intensity(percentage of control)

200100

Anti-CD44 Tf–647

300

Scrambled

Scrambled

Gal3siRNA1

Gal3siRNA1

Gal3siRNA2

Gal3siRNA2

∗∗∗∗

Gal3siRNA1

Gal3siRNA2

PP

MP

Anti-CD44 versus Tf–647 uptake

∗∗∗∗

Control

Control

a2 min

CD

44/G

al3–

Cy3

CD44 Gal3

Tf–4

88/G

al3–

Cy3

10 min

CD44 Gal3 CD44 Gal3

40 min

Tf–488 Gal3 Gal3Tf–488 Gal3Tf–488

+Gal3(μg ml–1)

Gal3siRNA3

ClathrinsiRNA2

Gal3/clathrinsiRNA

MEFWT

Figure 4 Endocytic function of Gal3. (a) Co-distribution analysis of Gal3 withCD44 or Tf. Cells were co-incubated for the indicated times at 37 ◦C with Gal3(red) and Tf or anti-CD44 antibodies (green). Gal3 co-localized strongly withCD44 at early times, and with Tf at later times. (b) Manders’ coefficientswere determined on experiments as in a. 12–15 cells per condition,means ± s.e.m., n=3 independent experiments. (c) CD44 endocytosis inGal3-depleted MEFs. Quantification of marker uptake (2min) in the indicatedconditions. Addition of exogenous Gal3 (1 µgml−1) to Gal3-depleted cellsrescued CD44 uptake only on cells with normal GSL levels. 8–24 cells percondition, means ± s.e.m., n=3 independent experiments. (d) Experimentas in c, in which increasing concentrations of recombinant Gal3 wereadded back to Gal3-depleted MEFs. 19–30 cells assessed from 5 fields percondition, means ± s.d., n=3 independent experiments. (e,f) Back-addition

of recombinant Gal3 to Gal3-deficient cells. Note that recombinant Gal3 at0.8 µgml−1 rescued endogenous staining levels. 36–44 cells assessed from4 fields per condition, means, n=2 independent experiments. (g) β1-integrinendocytosis experiment in the indicated depletion conditions. MEFs wereincubated for 10min with anti-β1-integrin antibodies. Note that both Gal3and clathrin contribute to β1-integrin uptake. 19–30 cells assessed from5 fields per condition, means ± s.d., n=3 independent experiments. Tfuptake was 92% inhibited (±3%) in the clathrin-depletion condition, means± s.d., n=3 independent experiments. (h) Fluid-phase HRP CLIC formation(2min HRP incubation with MEFs at 37 ◦C). Fluid-phase CLIC formation wasreduced in Gal3-depleted cells. 18–30 cells per condition, means ± s.e.m.,n=3 independent experiments. Statistical analysis in this figure: Student’sunpaired t-test, ∗P<0.05, ∗∗P<0.01. Scale bars, 10 µm.

Gal3 localizes to membrane invaginationsHRP-tagged Gal3 was often detected in plasma-membrane-connectedtubular invaginations (Fig. 6a, arrow). On average, one tubularinvagination was found for every 20.3± 3.5 µm of plasma membranecontour length (means ± s.e.m., n= 3 independent experiments,5–10 cells each), which is similar to other endocytic events45. To test

whether active cellular machinery such as molecular motors ortrafficking GTPases were strictly required for tubule formation, wedepleted cellular ATP. Under these conditions, Gal3–HRP was foundin patches at the plasma membrane (Fig. 6b, top) and in plasma-membrane-connected invaginations (middle and bottom). Tubulefrequency increased to one such structure for every 4.3 µm of plasma



ART ICLES

100

c Anti-CD44 uptakeFluorescence intensity(percentage of DMSO)

DMSO

Kifunensine

80604020

Deoxymanno-jirimycin

Swainsonine

∗∗

f Gal3Gal3

30 s30 s

Gal4Gal3 gh Gal4–HRPPM

Anti-HA

Anti-CD44 uptake

HA–CD44 WT HA–CD44 5xN-Glyc mt

Anti-HA

Anti-CD44 uptake

d

e Anti-CD44 uptakeFluorescence intensity

(percentage of HA–CD44 WT)

∗∗

20 100806040

HA–CD44WT

HA–CD445xN-Glyc mt

Carriers per cell section20151050

Tubules, rings (CLICs)Vesicles 80–120 nmVesicles 40–60 nm

i Gal4–HRP

Level of clustering L(r )0 ≥200100 15050

Deo

xym

anno

jirim

ycin

aC

ontr

ol

Control Deoxymanno-jirimycin

600

400

200

1,000

800

Mol

ecul

es (μ

m–2

)

CD44 molecule density

∗∗∗∗


0.015

0.010

0.005

Clu

ster

siz

e (μ

m–2

) ∗∗CD44 cluster size

b

0 0

Figure 5 Role of N-glycosylation in CD44 uptake. (a) Clustering analysisof CD44 in MEFs derived from single-molecule localizations obtainedwith dSTORM imaging. (b) The average density of CD44 molecules andCD44 cluster size decreased on treatment with 1-deoxymannojirimycin,demonstrating that complex type N-linked glycans were required for efficientCD44 clustering. Each symbol represents one image region; horizontallines indicate mean ± s.e.m., n=3 independent experiments. (c) CD44uptake was strongly inhibited in MEFs in which the biogenesis of complextype N-glycosylation was perturbed with the indicated inhibitors. 19–20cells per condition, means ± s.d., n=3 independent experiments. (d) CD44N-glycosylation site mutant. A human CD44 variant in which all 5N-glycosylation sites were mutated failed to be taken up into MEFs.(e) 17–19 cells assessed from 8 fields per condition in experiments asin d, means ± s.d., n=3 independent experiments. (f) Co-incubation of

TECs for 30 s with Gal3 (green) and Gal4 (red), followed by lactose washon ice to remove cell surface accessible material. Both proteins localizedto mostly separate very early uptake structures (see text for quantification).(g) Two differently labelled Gal3 molecules extensively co-localized underthe same conditions as in f, proving the specificity of the observations.(h) MEFs were incubated for 2min at 37 ◦C with Gal4–HRP,as described for Gal3–HRP in Fig. 1e. Arrowheads point tovesicular structures of different diameters, and arrows to tubularand ring-shaped CLICs. PM: plasma membrane. (i) Quantificationof experiments as in h showed that Gal4 was mostly localized to CLICs.Means ± s.e.m., n = 15 cells analysed in 1 experiment. Statisticalanalysis in this figure: Student’s unpaired t-test, ∗∗P<0.01, ∗∗∗∗P<0.0001.Scale bars: a (left), 6 µm; a (middle, right), 500nm; d,f,g, 10 µm;h, 200nm.

membrane contour length (means, n= 2 independent experiments5 cells each). Hence, scission of Gal3-containing tubules seemed to bean active process.

Electron microscope tomography revealed that these tubules hadbasket- and ring-shaped profiles (Fig. 6c, plasma membrane inred; Supplementary Video 3), demonstrating that invaginations withcharacteristic CLIC-likemorphology could formdirectly at the plasma

membrane. Actin cytoskeleton could clearly be seen in the vicinity ofthe invaginated Gal3 domains (Fig. 6c, blue), in agreement with itsrole in Gal3 endocytosis (Fig. 2b,c) and the formation of Gal3-CLICs(Fig. 2a). After GSL depletion, fewer tubular Gal3–HRP structureswere found on ATP-depleted cells (Fig. 6d), and quantificationshowed that tubule frequency had dropped to one such structurefor every 43.3 µm of plasma membrane contour length (means, n=2



ART ICLES

Gal3

2 min

Gal3

–ATP / +PPMP

Gal3

Gal3

α5 -integrin

β1 -integrin

e f

TfR

g h i

a

c

b

STxB

Gal3

Gal3

–ATP

d

Gal3

Gal3

CD

44

–ATP

Gal3

Figure 6 Morphologic analysis of Gal3-containing membrane invaginations.(a) MEFs were incubated for 2min at 37 ◦C with Gal3–HRP, and processedfor electron microscopy in the absence of ascorbic acid. Gal3–HRP waspresent in cell-surface-connected tubular invaginations (arrow). (b) Electronmicroscopy analysis on Gal3–HRP localization on ATP-depleted MEFs. TheHRP reaction product appeared in membrane patches (top), wide cisternae-like invaginations (middle), and narrow invaginations of different types(bottom). (c) Electron microscope tomogram of images as in the middlepanel of b. Plasma membrane (red), actin (blue), microtubules (orange) andinvaginations (yellow and green) were colour-coded. Note the basket-shaped

Gal3 structures with typical CLIC morphology. (d) Experiment as in b onPPMP-treated cells. No invaginations were observed, and Gal3–HRP labellingappeared diffuse. (e–i) Cargo co-distribution. Gal3 (red) was incubated withATP-depleted TECs (or Gb3-expressing HeLa cells for i), which were thenlabelled (green) for endogenous β1-integrin (e), α5-integrin (f), CD44 (g) ortransferrin receptor (TfR) (h). STxB (50nM) was co-incubated with Gal3 (i).Gal3 overlapped with the first three markers, but not with TfRor STxB (see text for quantifications). Representative results ofn=3 independent experiments with 10–12 cells each. Scale bars:a–d, 200nm; e–i, 5 µm.



ART ICLES

independent experiments 5 cells each). This result mirrored theinhibition of CD44 endocytosis (Fig. 1c) and Gal3-CLIC formation(Fig. 1f) under GSL-depletion conditions.

We next examined Gal3-CLIC cargoes on Gal3 tubular uptakestructures. As expected from Gal3 binding (Fig. 3a,b) and Gal3-mediated uptake (Fig. 4c,d,g), 65.6% (means, 196 tubules pooled from2 independent experiments), 61.3% (means, n= 191 tubules pooledfrom 2 independent experiments), and 53.6% (means, n=140 tubulespooled from 2 independent experiments) of tubular Gal3 structures(red) co-labelled with β1-integrin (Fig. 6e, green), α5-integrin(Fig. 6f, green), or CD44 (Fig. 6g, green), respectively. In contrast,only 4.4% (means, n = 216 tubules pooled from 2 independentexperiments) of Gal3 tubules (red) also labelled for Tf receptor(TfR; Fig. 6h, green). Gal3 tubules thus contained a specificcomplement of Gal3 binders and CLIC cargo proteins.

The B-subunit of the bacterial Shiga toxin (STxB) interacts with α-but not with β-galactosidic linkages to which Gal3 binds. STxB waspreviously shown to induce tubular uptake structures after bindingto the GSL globotriaosylceramide (Gb3; ref. 46). Here, we found thatSTxB (green) and Gal3 (red) localized to different tubule populationson the same cells (Fig. 6i). This finding demonstrated that distinctcurvature-generating environments were obtained when endocytosiswas driven by lectins with differential glycan-binding characteristics.

In conclusion, these experiments showed that Gal3 favouredthe GSL-dependent formation of plasma membrane invaginationscontaining specific Gal3 interacting proteins, whose processing byscission required active cellular machinery.

Membrane-bound Gal3 drives membrane bendingTo directly address the role of Gal3 in membrane bending, we turnedto a liposome reconstitution system. Giant unilamellar vesicles(GUVs) were prepared with a plasma-membrane-like lipid composi-tion containing cholesterol (20mol%), sphingomyelin (12mol%),1-palmitoyl-2-oleoylphosphatidyl choline (POPC, 59.9mol%),1-palmitoyl-2-oleoylphosphatidyl serine (POPS, 3mol%), a GSLmixture (5mol%, see Experimental Procedures), and 0.1mol%lissamine-rhodamine-B-PE for visualization. The GSL mixturecontained lipid species that were also found in the cells that wereused in the present study (Supplementary Tables 2 and 3). Gal3did not directly bind to these GSL-containing GUVs, in agreementwith the notion that Gal3 has low affinity for GSLs (ref. 29) andneeds glycosylated proteins for its recruitment onto membranes(Supplementary Figs 4b,c and 7d,e). Therefore, we His-tagged Gal3(Gal3–His) to force its binding to nickel-chelating lipid (3mol%)-containing GUV membranes, thus mimicking the binding of Gal3to cargo proteins at the plasma membrane. At early time points,Gal3–His was evenly distributed on NTA-containing GUVs (Fig. 7a,5min). At 15min, Gal3 was enriched locally (arrows and inset), andafter 30min 65.3% of GUVs showed tubular membrane invaginations(see Supplementary Table 4 for a summary of observations andquantification for all GUV experiments shown in this figure). 3.0± 1.6invaginations (n= 38 GUVs) were counted per equatorial plane ofGUVs to which Gal3 had bound, with an average length of1.9 ± 1 µm (n = 113 invaginations). Gal3 was enriched 2.5-fold(±0.9, n= 113) in these invaginations, when compared with thelimiting membrane.

Membrane tubules were at background levels when Gal3–His wasincubated with GUVs that did not contain GSLs (Fig. 7b), thatcontained 5mol% glucosylceramide, lactosylceramide, or Gb3, orwhen GSL-mixture-containing GUVs were incubated with a His-tagged GST–GFP control protein (Fig. 7c).

To disrupt carbohydrate-mediated interactions of Gal3 withGSLs, we incubated GUVs with Gal3–His in the presence oflactose as a competitive inhibitor. Under these conditions, Gal3–Hisfailed to cluster and form tubules on GSL-containing GUVs(Fig. 7d), demonstrating that Gal3 required direct interaction withthe carbohydrate part of GSLs to deform membranes. This importantconclusion was confirmed with a carbohydrate-binding-deficient His-tagged Gal3 mutant, Gal3-R186S, which did not induce invaginationson GSL-containing GUVs either (Fig. 7e).

Gal3 interacts with β-but not with α-galactosidic linkages. Tocharacterize the involvement of specific carbohydrate structures inthe tubulation process further, we treated GSL-containing GUVs withdifferent galactosidases. Incubation with β-galactosidase abolishedthe tubule-forming potential of Gal3–His, whereas α-galactosidasehad no effect (Fig. 7f and Supplementary Video 4), again pointingto a requirement of Gal3-carbohydrate interaction with GSLs formembrane bending. Taken together, membrane-bound Gal3 inducedinward-oriented tubulation on GUVs in a GSL-dependent manner.

Gal3 oligomerizes on membranesThe His-tagged Gal31Nter mutant is lacking the oligomerizationdomain and failed on cells to rescue CD44 uptake as efficiently as wild-type Gal3 (Fig. 4d). To analyse the contribution of oligomerization totubule formation, we used thismutant in ourGUV system. Gal31Nterhad a strongly reduced capacity to induce tubules (Fig. 7g), indicatingthat Gal3 clustering was important for membrane bending.

Direct evidence for Gal3 clustering came from experiments inwhich non-His-tagged Gal3 (which did not bind to GUVs on itsown) was recruited by Gal3–His onto GUVs (Fig. 7h). These dataclearly demonstrated the ability of Gal3 to interact with membrane-bound Gal3–His and consequently the propensity of Gal3 to undergooligomerization20,21. In contrast, non-His-tagged Gal31Nter failedto be recruited by Gal3–His (Fig. 7i), in complete agreement withthe notion that this protein has a reduced clustering capacity.The carbohydrate-binding mutant Gal3-R186S without a His-tagwas bound to GUVs by Gal3–His (Fig. 7j), indicating that sugarinteractions were not strictly required for oligomerization. On cells,this mutant failed to bind (Fig. 7k), but was internalized when co-incubated with wild-type Gal3 (Fig. 7l). Thus, we demonstratedthe propensity of Gal3 to undergo oligomerization on cell andmodel membranes.

DISCUSSIONOn the basis of the findings of this study, we propose a modelto explain the initiation of CLIC biogenesis (Fig. 8). MonomericGal3 is recruited to membranes by binding to glycosylated cargoproteins, such as CD44 and β1-integrin. Membrane-bound Gal3oligomerizes and acquires functional GSL interaction capacity. This islikely to involve an avidity effect based on multiple bond interactionsof binding sites on oligomeric lectins with several GSL molecules,and/or the creation of GSL-binding sites at interfaces between Gal3



ART ICLES

c 30 min

GS

T–G

FP–H

isb

d

+Lactose

30 minG

al3

–His

a 5 minG

al3

–His

15 min Zoom

200 nM 200 nM

f 30 min

Gal

3–H

is

+ β-galacto-sidase

30 min

Gal

3–H

is

+ α-galacto-sidase

Zoom

i

l Gal3 Gal3-R186S Merge

No GSL

30 min

Gal

3–H

is

j

Gal

3–H

is +

Gal

3 w

/o H

is

Gal3–His Gal3 w/o His ZoomMerge

g 30 min

Gal

3ΔN

ter–

His

200 nM

30 m

in

h

Gal

3–H

is +

Gal

3 w

/o H

is

Gal3–His Gal3 w/o His Merge

15 m

in

100 nM 100 nM 100 nM 100 nM

e

Gal

3-R

186S

–His

30 min

200 nM

Gal

3–H

is +

Gal

3-R

186S

w/o

His

ZoomMergeGal3–His Gal3-R186S

100 nM 100 nM

Gal3–His ΔNter w/o His

Gal

3–H

is +

ΔNte

r w

/o H

is

30 m

in

100 nM 100 nM

200 nM

k EEA1 Gal3-R186S

30 min Zoom

200 nM

40 nM 200 nM

Zoom

40 nM

Gal

3–H

is

Gal

3–H

is

Zoom

Figure 7 Gal3-driven membrane bending. (a–g) GUVs were prepared witha plasma-membrane-like lipid composition containing Ni-lipids and a mixof GSLs (except if indicated otherwise), incubated with Gal3–His (wild-type or mutants at 200nM, except if indicated otherwise) at 21 ◦C, andimaged at the equatorial plane by confocal microscopy. See SupplementaryTable 4 for quantifications of results. Gal3–His clustered (15min) andinduced invaginations (30min) (a). No invaginations formed with Gal3–Hisin the absence of GSLs (b), 200nM His-tagged GST–GFP (c), Gal3–Hisin the presence of 100mM lactose (d), the His-tagged Gal3 mutantR186S (e), Gal3–His on GSL-containing GUVs that were treated

with β-galactosidase (invaginations formed on GUVs treated withα-galactosidase as a control) (f), or His-tagged Gal31Nter mutant (g).(h–j) Non-His-tagged Gal3 (h), or Gal3-R186S (j) were recruited toGUVs only on co-incubation with Gal3–His, demonstrating that theseproteins oligomerized. In contrast, Gal31Nter was not co-recruited (i).(k) Gal3-R186S (red) did not associate with cells, labelled with the earlyendosomal marker protein EEA1 (green). (l) Co-incubation of Gal3-R186S(red) with wild-type Gal3 (green) led to the internalization of the mutant,strongly suggesting that Gal3 also oligomerized on cells. Scale bars: a–j, 3 µm;k,l, 5 µm.

monomers. Co-clustering of cargo proteins and GSLs then generatesmechanical strain to which the membrane responds by formingendocytic invaginations.

This model provides an integrative hypothesis of how cargoproteins are recruited and membranes bent in a process of clathrin-independent endocytosis. For CD44, endocytic uptake reliesextensively on the mechanism presented here. For β1-integrin,clathrin-dependent and -independent endocytic contributionshave previously been identified47, and our mechanism probablyexplains the clathrin-independent uptake. Indeed, β1-integrinfunctions have also been described to be reliant on N -glycosylation48

and GSL expression49, as one would expect from our model. An‘endocytic balance’ may thereby be set through an intricate interplaybetween different clustering mechanisms driven from the cytosolic(that is, coat-dependent) and extracellular (that is, galectin andglycosylation-dependent) sides of the plasma membrane.

In addition to CD44 and β1-integrin, a number of otherproteins are at the same time Gal3 binders and CLIC proteomicshits, including lysosomal-associated membrane protein-2 (Lamp-2),nicastrin subunit of γ-secretase, 4F2 heavy chain (CD98), the GPI-anchored protein CD109, mannose 6-phosphate receptor, collectin-12 and prosaposin. As these determinations were carried out on



ART ICLES

Gal-3–cargo complex Gal3 oligomerization and

GSL recruitment

CGal3Glycosylated

cargoGSL

N

C

Membrane bending andendocytic pit formation

Figure 8 Model for Gal3-driven biogenesis of CLICs. We propose thefollowing model to explain the Gal3-driven initiation of CLIC biogenesis.Monomeric Gal3 is recruited to membranes by binding to glycosylatedcargo proteins, such as CD44 and β1-integrin. Membrane-bound Gal3

oligomerizes and gains functional GSL binding capacity. Co-clusteringof cargo proteins and GSLs generates mechanical stress to which themembrane responds by forming endocytic invaginations. See discussion forfurther details.

different cell types (TECs versus MEFs, respectively), this overlap listmay represent an underestimation of candidate proteins whose uptakemight be mediated by Gal3.

On the basis of the fluid-phase uptake results, we concludethat Gal3 directly drives the biogenesis of a fraction of CLICs.At the same time, these experiments indicate that not all CLICformation is dependent on Gal3. Other galectins could be alternativebiochemical drivers, as exemplified here for Gal4. Whether additionalclasses of lectins or sphingolipid-interacting proteins also contributeremains to be addressed. Furthermore, actin-driven mechanisms ofglycolipid clustering, as described for GPI-anchored proteins50, couldin principle also generate endocytic nanoenvironments similar to theones described here.

Various studies have reported on stimulatory and inhibitory effectsof galectins on endocytosis23,51–55. Our finding that CD44 uptake isstimulated at serum-level concentrations of Gal3, but inhibited withGal3 concentrations that are several orders of magnitude above serumlevels provides an explanation for these apparently contradictory re-ports. Our model predicts that protein glycosylation and GSL expres-sion contribute to setting the domain boundary between regimes ofendocytic nanocluster formation (as described here), and the forma-tion of extended lattices leading to inhibition of internalization23.

In conclusion, we here propose a mechanism in which Gal3functions like an endocytic adaptor that drives the clathrin-independent biogenesis of endocytic pits by linking glycosylated cargoproteins and GSLs into compositionally defined nanoenvironments atthe plasma membrane. �

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe would like to thank the following people for help in experiments, providingmaterials or expertise: P. Bassereau, L. Cabanié, P. Chavrier, B. Hofmann, H. Ideo,

A. Raz, W. Römer, C. Schiff and T. Wollert. The facilities as well as scientific andtechnical assistance from staff in the Australian Microscopy and MicroanalysisFacility (AMMRF) at the Centre for Microscopy and Microanalysis at TheUniversity of Queensland, the Australian Cancer Research Foundation (ACRF)-Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology,the Biomedical Imaging Facility at UNSW and the PICT-IBiSA-Nikon ImagingCentre of Institut Curie with support from FRM (AAP ‘Grand Equipement’ 2011number DGE20111123020), Inca (Number 2011-1-Label-SALAMERO IC 4) andthe ‘CanNoli project’ supported by the DIM Canceropole-IdF (number 2012-2-EML-04) are acknowledged. This work was supported by grants from the NationalHealth and Medical Research Council of Australia (1037320, R.G.P. and K.G.;1045092, R.G.P., N.A. and M.T.H.), the Agence Nationale pour la Recherche (ANR-09-BLAN-283 and ANR-11 BSV2 014 03, L.J.), the Indo-French Centre for thePromotion of Advanced Science (project number 3803, L.J.), Marie Curie Actions—Networks for Initial Training (FP7-PEOPLE-2010-ITN, L.J.), European ResearchCouncil (project 340485, L.J.), fellowships from Association pour la Recherche surle Cancer (R.L.), Marie Curie International Reintegration Grant (FP7-RG-277078,C.W.), and Deutsche Forschungsgemeinschaft (U.B.). The L.J. team is a member ofLabex CelTisPhyBio.

AUTHOR CONTRIBUTIONSR.L., C.W. and U.B.: experimental design, method development and acquisition ofmajor data sets; U.B. and D.L.: protein MS; C.W.: guidance, and participated inmanuscript writing; M.T.H. and S.A.: electron microscopy—acquisition and dataanalysis; C.B. and K.G.: super-resolution microscopy; S.S. and A.S.: lipid analysis;N.A.: data analysis for electron microscope tomography; C.L.: technical supportand conceptual advice; V.C.: experimental support; R.G.P.: electron microscopy—study design, provided direction and guidance; L.J.: original conception of thestudy, provided direction and guidance, and wrote initial draft. All authors revisedthe manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2970Reprints and permissions information is available online at www.nature.com/reprints

1. Mayor, S. & Pagano, R. E. Pathways of clathrin-independent endocytosis. Nat. Rev.Mol. Cell. Biol. 8, 603–612 (2007).

2. Howes, M. T., Mayor, S. & Parton, R. G. Molecules, mechanisms, and cellular rolesof clathrin-independent endocytosis. Curr. Opin. Cell Biol. 22, 519–527 (2010).

3. Hansen, C. G. & Nichols, B. J. Molecular mechanisms of clathrin-independentendocytosis. J. Cell Sci. 122, 1713–1721 (2009).

4. Donaldson, J. G., Porat-Shliom, N. & Cohen, L. A. Clathrin-independent endocytosis:a unique platform for cell signaling and PM remodeling. Cell. Signal. 21, 1–6 (2009).

5. Sandvig, K., Pust, S., Skotland, T. & van Deurs, B. Clathrin-independent endocytosis:mechanisms and function. Curr. Opin. Cell Biol. 23, 413–420 (2011).

6. Lajoie, P. & Nabi, I. R. Lipid rafts, caveolae, and their endocytosis. Int. Rev. Cell.Mol. Biol. 282, 135–163 (2010).



http://www.nature.com/doifinder/10.1038/ncb2970





ART ICLES

7. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. GPI-anchored proteinsare delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 (2002).

8. Howes, M. T. et al. Clathrin-independent carriers form a high capacity endocyticsorting system at the leading edge of migrating cells. J. Cell Biol. 190,675–691 (2010).

9. Eyster, C. A. et al. Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis. Traffic 10, 590–599 (2009).

10. Naslavsky, N., Weigert, R. & Donaldson, J. G. Convergence of non-clathrin-and clathrin-derived endosomes involves Arf6 inactivation and changes inphosphoinositides. Mol. Biol. Cell 14, 417–431 (2003).

11. Lamaze, C. et al. Interleukin 2 receptors and detergent-resistant membrane domainsdefine a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 (2001).

12. Saint-Pol, A. et al. Clathrin adaptor epsinR is required for retrograde sorting on earlyendosomal membranes. Dev. Cell 6, 525–538 (2004).

13. Montesano, R., Roth, J., Robert, A. & Orci, L. Non-coated membrane invaginationsare involved in binding and internalization of cholera and tetanus toxins. Nature 296,651–653 (1982).

14. Kirkham, M. et al. Ultrastructural identification of uncoated caveolin-independentearly endocytic vehicles. J. Cell Biol. 168, 465–476 (2005).

15. Ewers, H. et al. GM1 structure determines SV40-induced membrane invaginationand infection. Nat. Cell Biol. 12, 11–18 (2010).

16. Leffler, H., Carlsson, S., Hedlund, M., Qian, Y. & Poirier, F. Introduction to galectins.Glycoconj. J. 19, 433–440 (2004).

17. Nakahara, S. & Raz, A. On the role of galectins in signal transduction. MethodsEnzymol. 417, 273–289 (2006).

18. Delacour, D., Koch, A. & Jacob, R. The role of galectins in protein trafficking. Traffic10, 1405–1413 (2009).

19. Boscher, C., Dennis, J. W. & Nabi, I. R. Glycosylation, galectins and cellular signaling.Curr. Opin. Cell Biol. 23, 383–392 (2011).

20. Ahmad, N. et al. Galectin-3 precipitates as a pentamer with synthetic multivalentcarbohydrates and forms heterogeneous cross-linked complexes. J. Biol. Chem. 279,10841–10847 (2004).

21. Lepur, A., Salomonsson, E., Nilsson, U. J. & Leffler, H. Ligand induced galectin-3self-association. J. Biol. Chem. 287, 21751–21756 (2012).

22. Dennis, J. W., Nabi, I. R. & Demetriou, M. Metabolism, cell surface organization, anddisease. Cell 139, 1229–1241 (2009).

23. Partridge, E. A. et al. Regulation of cytokine receptors by Golgi N-glycan processingand endocytosis. Science 306, 120–124 (2004).

24. Dumic, J., Dabelic, S. & Flogel, M. Galectin-3: an open-ended story. Biochim.Biophys. Acta 1760, 616–635 (2006).

25. Takenaka, Y., Fukumori, T. & Raz, A. Galectin-3 and metastasis. Glycoconj. J. 19,543–549 (2004).

26. Chiu, C. G. et al. Diagnostic utility of galectin-3 in thyroid cancer. Am. J. Pathol.176, 2067–2081 (2010).

27. Delacour, D. et al. Galectin-4 and sulfatides in apical membrane trafficking inenterocyte-like cells. J. Cell Biol. 169, 491–501 (2005).

28. Mishra, R., Grzybek, M., Niki, T., Hirashima, M. & Simons, K. Galectin-9 traffickingregulates apical-basal polarity in Madin-Darby canine kidney epithelial cells. Proc.Natl Acad. Sci. USA 107, 17633–17638 (2010).

29. Hirabayashi, J. et al. Oligosaccharide specificity of galectins: a search by frontalaffinity chromatography. Biochim. Biophys. Acta 1572, 232–254 (2002).

30. Collins, P. M., Bum-Erdene, K., Yu, X. & Blanchard, H. Galectin-3 interactions withglycosphingolipids. J. Mol. Biol. 426, 1439–1451 (2014).

31. Ravindran, M. S., Tanner, L. B. & Wenk, M. R. Sialic acid linkage inglycosphingolipids is a molecular correlate for trafficking and delivery of extracellularcargo. Traffic 14, 1182–1191 (2013).

32. Proia, R. L. Glycosphingolipid functions: insights from engineered mouse models.Phil. Trans. R. Soc. Lond. B 358, 879–883 (2003).

33. Furukawa, K., Tokuda, N., Okuda, T. & Tajima, O. Glycosphingolipids in engineeredmice: insights into function. Semin. Cell. Dev. Biol. 15, 389–396 (2004).

34. Kolter, T. & Sandhoff, K. Principles of lysosomal membrane digestion: stimulationof sphingolipid degradation by sphingolipid activator proteins and anionic lysosomallipids. Annu. Rev. Cell. Dev. Biol. 21, 81–103 (2005).

35. Ichikawa, S., Nakajo, N., Sakiyama, H. & Hirabayashi, Y. A mouse B16 melanomamutant deficient in glycolipids. Proc. Natl Acad. Sci. USA 91, 2703–2707 (1994).

36. Iurisci, I. et al. Concentrations of galectin-3 in the sera of normal controls and cancerpatients. Clin. Cancer Res. 6, 1389–1393 (2000).

37. Koca, S. S. et al. Serum galectin-3 level in systemic sclerosis. Clin. Rheumatol. 33,215–220 (2014).

38. Cederfur, C. et al. Different affinity of galectins for human serum glycoproteins:galectin-3 binds many protease inhibitors and acute phase proteins. Glycobiology18, 384–394 (2008).

39. Nieminen, J., Kuno, A., Hirabayashi, J. & Sato, S. Visualization of galectin-3oligomerization on the surface of neutrophils and endothelial cells using fluorescenceresonance energy transfer. J. Biol. Chem. 282, 1374–1383 (2007).

40. Ochieng, J., Leite-Browning, M. L. & Warfield, P. Regulation of cellular adhesion toextracellular matrix proteins by galectin-3. Biochem. Biophys. Res. Commun. 246,788–791 (1998).

41. English, N. M., Lesley, J. F. & Hyman, R. Site-specific de-N-glycosylation of CD44can activate hyaluronan binding, and CD44 activation states show distinct thresholddensities for hyaluronan binding. Cancer Res. 58, 3736–3742 (1998).

42. Ponta, H., Sherman, L. & Herrlich, P. A. CD44: from adhesion molecules to signallingregulators. Nat. Rev. Mol. Cell. Biol. 4, 33–45 (2003).

43. Velasco, S. et al. Neuronal galectin-4 is required for axon growth and for theorganization of axonal membrane L1 delivery and clustering. J. Neurochem. 125,49–62 (2013).

44. Ideo, H., Seko, A., Ohkura, T., Matta, K. L. & Yamashita, K. High-affinity bindingof recombinant human galectin-4 to SO(3(-) → 3Galβ1 → 3GalNAc pyranoside.Glycobiology 12, 199–208 (2002).

45. Griffiths, G., Back, R. & Marsh, M. A quantitative analysis of the endocytic pathwayin baby hamster kidney cells. J. Cell Biol. 109, 2703–2720 (1989).

46. Römer, W. et al. Shiga toxin induces tubular membrane invaginations for its uptakeinto cells. Nature 450, 670–675 (2007).

47. Margadant, C., Monsuur, H. N., Norman, J. C. & Sonnenberg, A. Mechanisms ofintegrin activation and trafficking. Curr. Opin. Cell Biol. 23, 607–614 (2011).

48. Janik, M. E., Litynska, A. & Vereecken, P. Cell migration-the role of integringlycosylation. Biochim. Biophys. Acta 1800, 545–555 (2011).

49. Wang, X. Q., Sun, P. & Paller, A. S. Inhibition of integrin-linked kinase/protein kinaseB/Akt signaling: mechanism for ganglioside-induced apoptosis. J. Biol. Chem. 276,44504–44511 (2001).

50. Goswami, D. et al. Nanoclusters of GPI-anchored proteins are formed by corticalactin-driven activity. Cell 135, 1085–1097 (2008).

51. Furtak, V., Hatcher, F. & Ochieng, J. Galectin-3 mediates the endocytosis of beta-1 integrins by breast carcinoma cells. Biochem. Biophys. Res. Commun. 289,845–850 (2001).

52. Fajka-Boja, R. et al. Co-localization of galectin-1 with GM1 ganglioside in thecourse of its clathrin- and raft-dependent endocytosis. Cell. Mol. Life Sci. 65,2586–2593 (2008).

53. Gao, X. et al. The two endocytic pathways mediated by the carbohydrate recognitiondomain and regulated by the collagen-like domain of galectin-3 in vascularendothelial cells. PLoS ONE 7, e52430 (2012).

54. Lepur, A. et al. Galectin-3 endocytosis by carbohydrate independent and dependentpathways in different macrophage like cell types. Biochim. Biophys. Acta 1820,804–818 (2012).

55. Zappelli, C., van der Zwaan, C., Thijssen-Timmer, D. C., Mertens, K. & Meijer, A.B. A novel role for galectin-8 as a mediator of coagulation factor V endocytosis bymegakaryocytes. J. Biol. Chem. 287, 8327–8335 (2012).



DOI: 10.1038/ncb2970 METHODS

METHODSMaterials. DNA constructs were verified by sequencing. The human Gal3construct (pGEX–6p–Gal3) was obtained from A. Raz56 (Detroit, USA). Alist of DNA constructs, primers and siRNAs is provided in SupplementaryTable 5. Antibodies: Gal3 (BD Pharmingen, number 556904, 1:500 and SantaCruz, sc-20157, 1:200), EEA1 (Santa Cruz Biotechnology, sc-6415, 1:100), TfR(Zymed Laboratories, clone H68.4, number 13-6890, 1:200), mouse CD44(Novus Biological, clone 5035-41.1D, NB100-65905 and Abcam, ab119863,10 µgml−1 (immunofluorescence), 0.2 µgml−1 for western blotting), humanCD44 (Novus Biological, clone Hermes-1, NBP2-22530, 1:500), α5-integrin(Millipore AB1928, 1:2,000 on methanol-fixed cells), β1-integrin (for westernblot: gift from P. Chavrier, Institute Curie, France; for immunofluorescence: BDPharmingen, clone 9EG7, #550531, 1:30), dynamin (Upstate, clone Hudy1, number05-319, 1:100), actin (Sigma-Aldrich, clone AC-74, A5316, 1:5000). Recombinantproteins (final concentration): CTxB–Alexa488 (0.5 µgml−1), CTxB–Alexa555(10 µgml−1, Invitrogen), Gal3–Cy3, Gal3–Alexa647, Gal3–Alexa488–His, Gal3-R186S–Cy3His (1–5 µgml−1), Gal3–HRP (10 µgml−1), Gal4–Cy3 (5 µgml−1),STxB–Cy3 (2 µgml−1, ref. 57), Tf–Alexa488/555/647 (10 µgml−1 Invitrogen).All lipids were purchased from Avanti Polar Lipids (Alabaster), except lipidstandards for shotgun lipidomics from Matreya LLC (Pleasant Gap). Otherchemicals were purchased from Sigma-Aldrich and were of ACS or LC-MS gradefor lipidomics.

Recombinant expression, purification and labelling. Gal3 and Gal4 proteinswere found by size-exclusion chromatography to be monomeric. Gal3–GST-containing plasmid (pGEX-6p-Gal3) was grown in Escherichia coli BL21 using 2xYTmedium. Protein expression was induced at D600 nm = 0.8 with 0.5mM IPTG at21 ◦C overnight. The cell pellet was resuspended in ice-cold PBS containing proteaseinhibitor cocktail and 5mM lactose. Bacteria were sonicated and adjusted to 1%Triton X-100 for further gentle agitation (30min, 4 ◦C). The supernatant (15,000g ,10min, 4 ◦C) was incubated with glutathione Sepharose (GE Healthcare) for 1 hat 4 ◦C. Beads were washed twice with PBS and PreScission cleavage buffer andincubated with PreScission protease (GE Healthcare) at 4 ◦C overnight (30 µl ofprotease and 470 µl of cleavage buffer per millilitre of beads). The supernatantcontaining Gal3 was dialysed against PBS twice to remove glutathione and lactose.SDS–PAGE and Coomassie staining showed purity above 90%.

For Cy3 labelling, Gal3 (2mgml−1) was dialysed against 5mM lactose in PBS.Amine-reactive (NHS-ester) Cy3 (GE Healthcare) or Alexa488/647 (Invitrogen)was added to the protein in a molar ratio of 9:1, and the reaction was incubatedfor 1 h at 21 ◦C with agitation. The mixture was purified using PD-10 columns(GE Healthcare).

His-tagged Gal3 (pHis2Parallel) and Gal3-R186S were expressed in Rossetta2-pLysS strains (Novagen) in LB media, supplemented with 60 µM IPTG at 20 ◦Covernight. 95% purity by Coomassie staining was achieved using Cobalt-resin(Pierce) affinity chromatography and gel filtration (Superdex75 16x60). Purificationbuffers were PBS at pH 7.4, or 0.2M bicarbonate buffer at pH 9.4 with 10%glycerol if subsequent coupling with HRP was planned. Alexa488 succinimidylester (Invitrogen) coupling to His-tagged Gal3 was performed as recommended byInvitrogen. If required, the 6xHis tag was removed by incubation with TEV protease(1 µg per 100 µg protein) at 4 ◦C overnight, and target proteins were subsequentpurified by FPLC-gel filtration.

Periodate-activated horseradish peroxidase (HRP; Pierce) coupling to His-taggedGal3was performed for 1 h at 21 ◦C in 0.2Mbicarbonate buffer at pH9.4, 10%glycerol at a molar ratio of 1:8. To separate HRP, Gal3 and Gal3–HRP, gel filtrationchromatography was performed using a Superdex75 10×30 column. Gal3–HRP in50% glycerol was snap-frozen for further usage.

Gal4–His (pQE-9, gift from H. Ideo, Tokyo Institute of Technology, Japan) wasgrown in M15 (pREP4) in LB media and precultured at 30 ◦C. Protein expressionwas induced with 10 µMIPTG at 16 ◦C overnight, bacterial pellets were resuspendedin 25mM HEPES at pH 7.4, 300mM NaCl, 10mM imidazole, 10% glycerol (bufferA), and sonicated. The lysate was adjusted to 5% ELUGENT (w/v, Merck), keptagitating for 30min at 4 ◦C, and cleared by centrifugation at 75,000g for 1 h. 95%purity by Coomassie staining was achieved using Cobalt-resin (Pierce) affinitychromatography (wash buffer: buffer A + 1% ELUGENT; elution buffer: 25mMHEPES at pH 7.4, 300mMNaCl, 20mM trehalose, 0.2% (w/v) PEG8000+ 300mMimidazole) and gel filtration (Superdex75 16×60). Fluorescence labelling and snap-freezing were performed in elution buffer without imidazole. Gal4 was coupled at4 ◦C overnight to periodate-activated HRP in 0.2M bicarbonate buffer at pH 9.4,300mMNaCl, 10% glycerol, 10mM lactose, 20mM trehalose, 0.2% (w/v) PEG8000,and purified by gel filtration in the same buffer without lactose.

GST–GFP–His (pGEX4T-1) preferentially exists as a dimer and was expressedovernight at 25 ◦C in Rossetta2-pLysS strains in LBmedia, supplementedwith 10 µMIPTG. 95% purity by Coomassie staining was achieved using Cobalt-resin affinitychromatography and gel filtration (Superdex75 16x60) in PBS.

Cell culture, transfection and treatments. TECs, HeLa cells and MEFs werecultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10%fetal bovine serum (FBS), 1mMsodiumpyruvate, and 2mMglutamine (Invitrogen).Wild-type MEFs were immortalized as previously described58. MEB4, GM95 andCG1 cells were cultured in the same media but with 5% FBS. HeLa cells weretransfected 24 h after plating with Fugene6 (Roche). TECs were transfected byelectroporation using the ElectroBuffer kit (Cell Projects) and Gene Pulser-II(BioRad) at 200V and 975mF with 4mm cuvettes (DNA-cell volume: 250 µl)according to the manufacturer’s instructions.

GSL depletion of cells was achieved by incubation with 5 µM D,L-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, Enzo Life Sciences)for 6–14 days (MEFs and HeLa cells: 6 days, TECs: 14 days). ATP depletion wasinitiated 30min before incubation with Gal3. Cells were incubated for 30minat 37 ◦C in ATP-depletion media composed of PBS++ (PBS + 0.5mM CaCl2,0.5mM MgCl2), 10mM 2-deoxy-D-glucose, and 10mM NaN3. Cells were kept inATP-depletion media for subsequent manipulation. For competition experimentswith free sugars, cells were incubated for 30min in serum-free media beforetreatment with 0.1M sucrose or 0.1M lactose for 20min. The sugars were keptthroughout subsequent incubation steps. Cells were pre-treated with latrunculin A(10–20min, 1 µMonMEFs, 2 µMonTECs), 12.5 µgml−1 tunicamycin (24 h), 10 µMdynoles 31-2 or 34-2 (20min), 10 µgml−1 kifunensine (48 h, 2% FBS), 90 µgml−11-deoxymannojirimycin (48 h, 2% FBS), or 2µgml−1 swaionsonine (48 h, 2% FBS)before endocytosis or binding assays. Depletion ofGal3 and/or clathrinwas achievedafter transfection of siRNA using HiPerfect, according to the manufacturer’sinstructions, and incubation for 48 to 72 h (Supplementary Table 3). In the case ofGal3 and GSL depletion, treatment with PPMP was initiated 3 days before siRNAtransfection. Inhibitors were present throughout subsequent manipulation.

Immunofluorescence. MEFs, TECs or HeLa cells were washed with PBS++ twiceand incubated with Gal3–Cy3, CTxB–Alexa488/555, and/or Tf–Alexa647 for theindicated periods of time in serum-freemediumon ice or at 37 ◦C.Unboundproteinswere removed by two washes with PBS++. Where indicated, cell-surface-exposedmarkers were removed by 3× 30 s washes with ice-cold 0.5M glycine at pH 2.2,including in some cases 200mM lactose. Cells were fixed with 4% PFA (or −20 ◦Cmethanol for α5-integrin), permeabilized and labelled with indicated primary andsecondary antibodies. Samples were imaged by confocal microscopy with ×60or ×40 oil immersion objectives. For quantification, images were backgroundsubtracted, cellular regions were marked, and the total fluorescence intensity wasmeasured in these regions using ImageJ. Intensities were normalized separately tothe untreated control sample of each marker. Co-localization between markers wascalculated as Manders’ coefficients using ImageJ’s co-localization plugin (NIH).

Endocytosis assays. For immunofluorescence, MEFs, TECs or HeLa cells wereincubated in fresh growth media for 30min and subsequently with anti-CD44antibodies (10 µgml−1), anti-β1-integrin antibodies (1 µgml−1) and/or Tf–Alexa633(10 µgml−1) for the indicated periods of time in growth medium on ice or at 37 ◦C.Cell-surface-exposed markers were removed by 3× 30 s washes with ice-cold 0.5Mglycine at pH 2.2 (for quantification of endocytosis), or cells were left untreated(for quantification of surface levels). Cells were then washed, fixed (2% PFA for60min), permeabilized (1mgml−1 saponin, 10min), blocked (3× 10min in 0.2%BSA, 0.2% fish skin gelatine in PBS), and labelled with fluorophore-conjugatedsecondary antibodies. Samples were imaged and total fluorescence intensities werequantified as described above. In the case of HA–CD44 WT and HA–CD44 5xN-Glyc mt endocytosis, we expressed the human constructs in MEFs and used thehuman-specific anti-CD44 antibody (clone Hermes-1) for uptake experiments.

For biochemistry, indicated cell lines were plated on coverslips in growth medi-um containing 5% FBS to reach 80% confluence. Cells were placed in fresh growthmedia for 30min. Coverslips were incubated for 10min at 37 ◦C upside-downwith anti-CD44–FITC antibody (10 µgml−1) in growth medium containing 15mMHEPES at pH 7.3. Cell-surface-exposed antibodies were removed by 3× 30 s washeswith ice-cold 0.5M glycine at pH 2.2. Cells were washed, collected using trypsin–EDTA, and kept on ice after addition of protease inhibitors. The cell pellet was re-suspended in non-reducing sample buffer, boiled and analysed by SDS–PAGE. Anti-CD44–FITC antibody was detected using the ChemiDoc Gel imaging system (Bio-Rad). Fluorescent bands were background subtracted and quantified using ImageJ.

dSTORM imaging and data processing. See Supplementary Note 1.

Electronmicroscopy.Cells were pulsed for 2min at 37 ◦Cwith 10mgml−1 HRP or10 µgml−1 Gal3–HRP, cooled in ice-cold CO2-independentmedia before incubationwith ice-cold 10mgml−1 3,3′-diaminobenzidine (DAB) with or without 50mMascorbic acid for 10min, followed by a second wash in DAB, 0.012% H2O2 withor without ascorbic acid for 20min. Cells were washed 3× in CO2-independentmedia before fixation in 2.5% glutaraldehyde (ProSciTech). For ATP depletion

NATURE CELL BIOLOGY


METHODS DOI: 10.1038/ncb2970

experiments, cells were fixed before DAB reaction. Fixed samples were contrastedwith 1% osmium tetroxide (ProSciTech), 2% uranyl acetate (ProSciTech) and weredehydrated in ethanol before embedding in LX-112 epoxide resin (ProSciTech).For quantification of HRP-positive carriers per cell, samples were sectioned inan ultramicrotome (Leica), and 20–25 cells per condition analysed across twoindependent experiments. HRP-positive carriers were grouped into tubular/ring-shaped elements (CLICs), clathrin-coated vesicles (80–120 nm diameter), andcaveolae (40–60 nm diameter).

For electron microscope tomography, 280 nm sections were cut and dual-axistilt-series captured from ± 60◦ at 2 ◦ increments on a Tecnai F30 microscope(FEI Company) with an accelerating voltage of 300 keV. Tilt-series were imagedusing a Direct Electron LC-1100 4K by 4K camera at a binning of 2, usingthe microscope control program serialEM. Reconstructions were completed usingweighted back-projection in the software program IMOD. Segmentation analysiswas performed as described previously59, with the exception that membranes andcytoskeleton were manually segmented for each 5–10 slices and auto-interpolatedbetween manually inserted contours, using the interpolator function in IMOD.

Proteinase K treatment. TECs were washed with 20mM HEPES at pH 7.4,150mM NaCl, 5mM CaCl2 and incubated for 5min at 37 ◦C in the same buffersupplemented with 50 µgml−1 proteinase K (Ambion). Proteinase digestion wasquenched by one wash with ice-cold PBS and twice with PBS containing 2mMphenylmethylsulphonyl fluoride and 2mMEGTA. Protease inhibitors were removedby two washes with 20mM HEPES at pH 7.3, 75mM NaCl, 5mM KCl, 0.5mMMgCl2, 0.5mM CaCl2, 0.45mgml−1 glucose, 0.45mgml−1 albumin. Cells were kepton ice for 20min for plasmamembrane binding ofGal3 andCTxB in the same buffer,washed with ice-cold PBS, fixed in 4% PFA, 0.2% glutaraldehyde for 10min on ice,and for 20min at 21 ◦C.

Mass spectrometry and pulldown. TECs were washed with PBS++ and incubatedin lactose buffer (20mM HEPES at pH 7.2, 200mM lactose, 45mM NaCl, 5mMKCl, 1mMMgCl2, 1mM CaCl2) for 10min at 4 ◦C to remove endogenous, surface-bound Gal3. Cells were extensively washed with PBS++ and subsequently incubatedin PBS++ (negative control) or in PBS++ containing 2 µgml−1 Gal3–His (45min,4 ◦C). After rinsing twice with PBS, cells were lysed in PBS supplemented with20mM imidazole, 0.5% Triton X-100, 0.5% NP-40 and protease inhibitor cocktail(lysis buffer). Cleared lysates (16,100g , 10min, 4 ◦C) were incubated with NiNTA-Sepharose (Qiagen) (1 h, 4 ◦C) and washed with lysis buffer (without proteaseinhibitors). Gal3 binding partners were eluted with PBS + 200mM lactose andfurther processed for analysis by mass spectrometry or SDS–PAGE. For proteindeglycosylation, eluates were adjusted to 10mM βmercaptoethanol and 0.02% SDS,denatured at 95 ◦C for 5min and after cooling incubated with 42Uml−1 PNGase Ffor 2 h at 37 ◦C.

Proteins precipitated from 10mg total proteinweremigrated on SDS–PAGE gels.Excised gel slices were washed and proteins were reduced with 10mM dithiothreitolbefore alkylation with 55mM iodoacetamide. After washing and shrinking of thegel pieces with 100% acetonitrile, in-gel digestion was performed using trypsin(Sequencing GradeModified) overnight in 25mMammonium bicarbonate at 30 ◦C.We achieved peptide concentration and separation using an actively split capillaryhigh-performance liquid chromatography (HPCL) system (Ultimate 3000 system)connected to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Themass spectrometer was set to acquire a single MS scan followed by up to five data-dependent scans (dynamic exclusion repeat count of 1, repeat duration of 30 s,exclusion duration of 300 s and lock-mass optionwas enabled). The resulting spectrawere analysed using theMascot Software createdwith ProteomeDiscoverer (version:1.2.0.92, Thermo Scientific) using the NCBInrMusmusculus (housemouse) ProteinDatabase (2012 04 02, 144149 sequences). The resulting Mascot result files werefurther processed using myProMS (ref. 60). The estimated false discovery rate ofall peptide and protein identifications was less than 1%, by automatically filtering onpeptide length, mass error and Mascot score of all peptide identifications.

Shotgun lipidomics. For lipid extraction, to 1ml cell suspension (equivalent to6.5mgml−1 and 6.9mgml−1 of the total protein content for TECs and MEF cells,respectively) 10ml of chloroform/methanol/water (4:8:3, v/v/v; ref. 61) was added.The mixture was extracted for 30min at 21 ◦C, centrifuged, and the supernatanttransferred to a glass vial. The pellet was re-extracted with another 5ml of thechloroform/methanol/water mixture. The combined supernatants were dried in avacuum centrifuge (Jouan RC1022, Thermo Fisher Scientific).

Two-phase partitioning was performed as described previously62 withminormodifications. Four millilitres of di-isopropyl ether/1-butanol (6:4, v/v) was addedto the dried extract, vortexed and sonicated, followed by adding 2ml 50mM NaCl(aq.). The mixture was again vortexed and sonicated for several minutes and thencentrifuged to separate the phases. The upper organic phase was transferred to aglass vial and saved for subsequent alkaline hydrolysis. The lower aqueous phase was

re-extracted with 4ml of di-isopropyl ether/1-butanol (6:4, v/v), vortexed, sonicatedfor several minutes and centrifuged. The organic phase was removed and saved forsubsequent alkaline hydrolysis. Aqueous phases from both extraction steps werepooled, dried in a vacuum centrifuge, and re-dissolved in 1ml methanol/water(1:1, v/v) with vortexing and sonication.

For recovery of gangliosides by solid phase extraction, aqueous phase collectedafter two-phase partitioning was cleaned-up by solid-phase extraction63. SupelcleanLC-18 SPE columns (Supelco) were successively preconditioned with 10mlmethanol, 20ml chloroform/methanol (2:1, v/v), 10ml methanol, and 20mlmethanol/water (1:1, v/v). The aqueous phase was loaded onto the column. Thecolumn was washed with 40ml H2O, and gangliosides were eluted with 2mlmethanol and 10ml chloroform/methanol (1:1, v/v) mixture. The gangliosidefraction was dried and re-dissolved in 150 µl of chloroform/methanol (1:1, v/v).

For alkaline hydrolysis of the organic phase, 1ml of 0.2M NaOH in methanolwas added to the dried sample, incubated at 30 ◦C for 120min and neutralized byadding 8 µl of acetic acid. Chloroform (1ml) and water (2ml) were added and themixture vortexed for 1 h at 21 ◦C. The lower organic phase was transferred to a glassvial, dried in a vacuum centrifuge and re-dissolved in 150 µl chloroform/methanol(2:1, v/v).

Thin-layer chromatography was performed on Silica gel 60 F254 plates(20×20 cm; Sigma-Aldrich) in chloroform/methanol/CaCl2 (65:35:8) and stainedwith 20% H2SO4.

For lipid analysis by shotgun mass spectrometry, 2 µl aliquots of the cell extractsprepared as described above were 160-fold diluted with 7.5mM ammonium acetatein 2-propanol for positive-mode analyses and 20-fold with 0.05% triethylamine inmethanol for negative-mode analyses. The total ganglioside extract (Avanti PolarLipids) used as a reference was dissolved in chloroform/methanol/water (65:35:8,v/v) at the concentration of 1mgml−1 and diluted 100-fold for analyses in positiveand negative modes.

Shotgun lipidomics analyses were performed on a Q Exactive mass spectrometer(Thermo Fisher Scientific) equipped with a robotic nanoflow ion source TriVersaNanoMate (Advion BioSciences). FT MS spectra were acquired for 1min withinthe range of m/z 450–1750 at the target mass resolution of R= 140,000 (full-width at half-maximum at m/z 200) and automated gain control (AGC) of1×106. Lipid species were identified using LipidXplorer software64 by their m/zdetermined in MS survey scans with better than 3 ppm accuracy and a signal tonoise ratio above the value of 3.0. The molecular fragmentation query language(MFQL) queries required for the lipid identification are available at the LipidXplorerwiki site at https://wiki.mpi-cbg.de/lipidx/Main_Page. Lipid loadings were adjustedaccording to the total protein content determined by the BCA protein assay (Pierce).Species of the following lipid classes were monitored: sphingomyelins, ceramides,hexosylceramides, di-hexosylceramides, GA2, GA1/Gb4 (were not distinguishedbecause they have identical masses), Gb3, GD1, GM3, GM2, GM1, GT1, GD3, GD2,GT3, GT2, GQ1, GP1, sulphatides, Forssman antigen.

To confirm the identity of thin-layer chromatography bands, they were scrapedfrom the unstained plate and extractedwith 500 µl of chloroform/methanol (2:1, v/v)for 20min at room temperature. After centrifugation for 15min at 4 ◦C thesupernatant was carefully collected, transferred into a glass vial and dried under anitrogen stream. The dried sample was re-suspended in 100 µl chloroform/methanol(2:1, v/v) and analysed as described above.

Model membrane studies. GUVs were prepared essentially as describedpreviously65. A thin film of lipid mixtures (2mgml−1) containing POPC(56.9mol%), cholesterol (20mol%), sphingomyelin (12mol%), POPS (3mol%),GSL-mix (5mol%), Ni-NTA-DOGS (3mol%) and lissamine-rhodamine-PE(0.1mol%) was applied to glass slides covered with indium-tin oxide (DeltaTechnologies) and placed into a custom-made Teflon chamber. The GSLmix was atotal ganglioside extract from porcine brain and contained the trisialogangliosideGT1b (44.5%), disialogangliosides GD1a (33%), GD1b (8%) and GD1bL (7%),and monosialogangliosides GM1 (6.5%), GM2 (0.66%) and GM3 (0.33%). Theglass slides were separated by a 2mm thin sucrose solution (600mM). An electrica.c. field (0.389VRMS, 10Hz) was applied for 2.5 h at 65 ◦C following the previouslydescribed GUV electroformation protocol66. Liposomes were collected after coolingto 21 ◦C and used immediately. A 200 µl observation chamber (Lab-Tek chambered#1.0 borosilicate) was coated using 2mgml−1 BSA to avoid distortion of GUVs oncontact with the chamber and rinsed with imaging buffer (10mM HEPES at pH7.2, 590mM glucose). One hundred microlitres of GUVs in 600mM sucrose wasmixed with 100 µl of Gal3–His, Gal3-R186S–His, Gal3-R186S or GFP–GST–Hisin imaging buffer to yield 200 nM (that is, 5 µgml−1 of Gal3–His, respectively)final concentrations in 300mM sucrose. The buffer and proteins added to GUVsmatched the osmolarity of the GUV solution. The solution was stirred to accelerateprotein distribution and binding. A buffer control was performed for eachexperiment. This control was treated exactly the same as the actual experiment;however, only buffer and no recombinant protein was added. Images were taken in

NATURE CELL BIOLOGY


https://wiki.mpi-cbg.de/lipidx/Main_Page

DOI: 10.1038/ncb2970 METHODS

multi-tracking mode on a Zeiss LSM510 laser-scanning confocal microscope witha ×63/1.4NA Plan Apochromat objective and a 488/543 dichroic mirror, and ona Leica SP8 microscope for movie acquisitions. GFP/Alexa-488 dyes were excitedusing the 488 nm argon laser, and rhodamine was excited with a 543 nm He/Nelaser. GFP and Alexa488 emission was collected with 505–530 nm, and rhodamineemission with 560–615 nm bandpass filters. The pinholes for each channel wereset for an approximate 0.8–1.1 µm optical slice. To prevent light-induced artefacts,images were acquired with an approximate interval of 5min for each condition.For lipid mixtures without GSLs or without Ni-NTA-DOGS, both lipids werereplaced by POPC. Gal3 sorting on GUV tubules was calculated using fluorescenceintensities (Int) of Alexa488–Gal3 normalized by the mean intensity of lissamine-rhodamine-PE according to ref. 67: Sorting (S)= (Int(Gal3tubule)/Int(Gal3membrane))/(Int(PE-rhodaminetubule)/Int(PE-rhodaminemembrane)). Galactosidase treatments with10Uml−1 of α-galactosidase (Sigma G8507, green coffee beans) or β-galactosidase(Sigma G5160, Aspergillus oryzae) of GUVs containing total GSL-mixture wasperformed for 60min at 37 ◦C before Gal3 incubation by mixing GUVs in sucrosewith the digestion/imaging buffer (final concentration: 10mM HEPES at pH 6.5,5mM CaCl2, 300mM sucrose, 290mM glucose).

56. Ochieng, J. et al. Structure-function relationship of a recombinant humangalactoside-binding protein. Biochemistry 32, 4455–4460 (1993).

57. Johannes, L., Tenza, D., Antony, C. & Goud, B. Retrograde transport of KDEL-bearingB-fragment of Shiga toxin. J. Biol. Chem. 272, 19554–19561 (1997).

58. Kalia, M. et al. Arf6-independent GPI-anchored protein-enriched early endosomalcompartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3’-kinase-dependent machinery. Mol. Biol. Cell 17, 3689–3704 (2006).

59. Richter, T. et al. High-resolution 3D quantitative analysis of caveolar ultrastructureand caveola-cytoskeleton interactions. Traffic 9, 893–909 (2008).

60. Poullet, P., Carpentier, S. & Barillot, E. myProMS, a web server for managementand validation of mass spectrometry-based proteomic data. Proteomics 7,2553–2556 (2007).

61. Svennerholm, L. & Fredman, P. A procedure for the quantitative isolation of braingangliosides. Biochim. Biophys. Acta 617, 97–109 (1980).

62. Ladisch, S. & Gillard, B. A solvent partition method for microscale gangliosidepurification. Anal. Biochem. 146, 220–231 (1985).

63. Senn, H. J., Orth, M., Fitzke, E., Wieland, H. & Gerok, W. Gangliosides in normalhuman serum. Concentration, pattern and transport by lipoproteins. Eur. J. Biochem.181, 657–662 (1989).

64. Herzog, R. et al. A novel informatics concept for high-throughput shotgunlipidomics based on the molecular fragmentation query language. Genome Biol. 12,R8 (2011).

65. Wollert, T., Wunder, C., Lippincott-Schwartz, J. & Hurley, J. H. Membrane scissionby the ESCRT-III complex. Nature 458, 172–177 (2009).

66. Angelova, M. I. & Dimitrov, D. S. Liposome electroformation. Faraday Discuss. Chem.Soc. 81, 303–311 (1986).

67. Safouane, M. et al. Lipid cosorting mediated by Shiga toxin induced tubulation.Traffic 11, 1519–1529 (2010).

NATURE CELL BIOLOGY


S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb2970

Supplementary Figure 1

2 min

PPMP

Control

10 min

PPMP

Control c

CD44

GM95MEB4 CG1

e

CD44CTxB-555

Fluo

resc

ence

inte

nsity

(% o

f con

trol

)

80

60

40

20

120

100

**Control PPMP

CD44 CTxB

Surface

PPMP

Control

b

f 10 μg/ml 1 μg/ml

500 ng/ml

100 ng/ml 10 ng/ml

1 ng/ml

Gal3

-HRP

g Control PPMP

d

GM95MEB4

Fluo

resc

ence

inte

nsity

(% o

f MEB

4)

80

60

40

20

120

100

GM95

CD44

MEB4

PPMPCtrl.

Fluo

resc

ence

inte

nsity

(%of

con

trol

)

80

60

40

20

120

100

GM95MEB4 CG1

Fluo

resc

ence

inte

nsity

(% o

f MEB

4)

80

60

40

20

120

100

Anti-CD44FITC

** ****

CG1

CD44 surface levels

CG1

Gal3

-HRP

Anti-CD44-FITC uptake

CD44 Tf-555 CD44 Tf-555

CD44 Tf-555 CD44 Tf-555

CD44 CD44

CD44 CD44

a

Control Control

PPMP PPMP

Surface levels

PPMPCtrl.

Supplementary Figure 1 GSLs are required for CD44 uptake. a, Control or PPMP-treated MEFs were incubated at 37°C with anti-CD44 antibodies (green) and Tf (red) (both at 10 µg*ml-1) for 2 or 10 min, and cell surface exposed material was removed by acid washes. CD44 uptake was strongly inhibited in PPMP-treated cells. Quantification is shown in Fig. 1c. b, The indicated types of cells were incubated for 2 min with anti-CD44 antibodies, and cell surface exposed material was removed by acid washes. CD44 uptake was strongly inhibited in GSL-deficient GM95 cells. Quantification is shown in Fig. 1d. c, Control or PPMP-treated MEFs were incubated on ice with anti-CD44 antibodies (green) and CTxB (red). Quantification on 15-20 cells per condition (means ± S.E.M., n=3 independent experiments). PPMP treatment significantly reduced CTxB binding, but not that of anti-CD44 antibody. d, The indicated types of cells were incubated as in (c). Similar CD44 surface levels were observed on all cells. Quantification on 38-49 cells assessed from 4 fields (means, n=2 independent experiments). e, Biochemical CD44 uptake assay. Fluorescently labelled anti-CD44 antibodies at 10 µg/ml were incubated for 10 min at 37°C with MEFs in the indicated conditions (± PPMP), or with MEB4, GM95, or CG1 cells.

After acid washes, the cells were lysed and lysates analysed by SDS-PAGE and fluorescence detection. Means ± S.D are shown, n=4 (upper graph) and n=5 (lower graph) independent experiments. Note that uptake efficiency was strongly dependent on GSL expression. The main GSL, GM3, was 35-fold less expressed in CG1 cells than in MEB4 cells, likely explaining the partial rescue of the CD44 uptake phenotype. A representative gel is shown for the ±PPMP conditions. f, Dose-response of Gal3-HRP CLIC formation. MEFs were incubated for 2 min at 37°C with the indicated concentrations of Gal3-HRP. The diaminobenzidine reaction for imaging HRP was performed on ice in the presence of ascorbic acid, and cells were processed for EM. Gal3-CLIC (tubules/rings) were observed with Gal3-HRP concentrations as low as 10 ng*ml-1. g, Control or PPMP-treated TECs were incubated for 2 min at 37°C with Gal3-HRP. The diaminobenzidine reaction for imaging HRP was performed on ice in the presence of ascorbic acid, and cells were processed for EM. Gal3-CLIC (tubules/rings) formation was significantly inhibited on PPMP-treated cells. Quantification is shown in Fig. 1f. Statistical analysis in this figure: Student, unpaired t-test. ** p<0.01. Scale bars = 10 µm in a-d, and 200 nm in f,g.


http://www.nature.com/doifinder/10.1038/ncbxxxx


2 WWW.NATURE.COM/NATURECELLBIOLOGY

SupplementaryFigure 2

d f

g

Control PPMP

300

200

100

500

400

Mol

ecul

es (p

er μ

m )2

Control PPMP

5

15

10

Clu

ster

s (p

er μ

m )2

Gal3 cluster density

Control PPMP

Gal3 cluster radius

80

70

60

100

90

50C

lust

er ra

dius

(nm

)

Control PPMP

Gal3 cluster size

0.01

0.03

0.02

Clu

ster

siz

e (μ

m )2

Con

trol

a

PPM

P

b c

0 50 100 150 >200Level of clustering L(r )

i j kControl cells

+ Gal3-Alexa647 PPMP-cells + Gal3-Alexa647 Gal3-Alexa647 only

Off-Gap (frames)0

Num

ber o

f mol

ecul

es

10 20 4030 50 7060 9080 100Off-Gap (frames)

10 20 4030 50 7060 9080 100

Num

ber o

f mol

ecul

es

Off-Gap (frames)

0

Num

ber o

f mol

ecul

es

10 20 4030 50 7060 9080 1000

0

00

e Average Gal3 molecule density

1.5*106

1.0*106

0.5*106

6*105

4*105

2*105

1.5*106

1.0*106

0.5*106

Gal3 clustering and control

60

30

0

90

Max

of L

(r) —

r

h

120

Control PPMP Clustercontrol

****

Supplementary Figure 2 dSTORM imaging and cluster analysis of Gal3-Alexa647 in control and PPMP-treated HeLa cells. a, dSTORM images of Gal3-Alexa647 in HeLa cells. Cells were pre-treated or not with PPMP, and incubated with 4 µg*ml-1 of Gal3-Alexa647 for 20 min, fixed and imaged under total internal reflection fluorescence (TIRF) illumination. b, Color-coded cluster maps retrieved from local point pattern analysis of regions (red squares in (a)). Colour indicates level of clustering, L(r), with low to high clustering coloured blue to red. c, Maps of Gal3-Alexa647 clusters and molecules inside clusters after applying a threshold to color-coded maps shown in (b). d-g, Density of Gal3-Alexa647 molecules (d), density of Gal3-Alexa647 clusters (e), radii of Gal3-Alexa647 clusters in nm (f), area covered by Gal3-Alexa647 clusters (g) were obtained from threshold cluster maps. Means ± S.E.M from 10-20 cells, n=3 independent experiments

for each panel. h-k, Analysis for the effects of photoblinking. Max of L(r)-r values (h) of Gal3-Alexa647 molecules adhered to coverslips under cell-free conditions compared to corresponding data of Gal3-Alexa647 on control and PPMP-treated HeLa cells, shown in Fig. 1g,h. To correct for repeated excitation of the same fluorophores, the number of localized Galectin3-Alexa647 molecules in control (i) and PPMP-treated (j) HeLa cells as well as Alexa647-Gal3 molecules adhered to coverslips (k) was plotted versus the off-gap. Data (round symbols) was fitted (solid line) to equation 4 (see Supplementary Note 1). The off-gap threshold was at 40 frames, illustrating that in three examples, >97.5% of over-counted molecules were removed. i-k are representative experiments, n=3 independent experiments with 10-20 cells examined per experiment. Scale bars: a = 6 µm, b,c = 500 nm. Student, unpaired t-test, ** p<0.01; *** p<0.001; **** p<0.0001.




Control DynK44A-GFP

Fluorescence intensity(% of control)

Tf

Gal3

***

10080604020

Control EH2-1-GFP

Tf

Gal3

***

10080604020

*


c (HeLa)

d (TEC)

b (TEC)


a (HeLa)

Tf Gal3

***Fl

uore

scen

ce in

tens

ity(%

of c

ontro

l) 100

604020

140120

80

Control EH2-1-GFP

Tf Gal3

Fluo

resc

ence

inte

nsity

(% o

f con

trol)

***

Control DynK44A-GFP

100

604020

140120

80

EH2-1-GFP Gal3 Tf

15 min

TfDyn2K44A-GFP

Dyn2K44A-GFP Gal3

15 min

15 min

TfDyn2K44A-GFP Gal3

15 min

TfEH2-1-GFP

15 minGal3EH2-1-GFP

15 min

Supplementary Figure 3 Gal3 endocytosis under perturbation conditions. a,b, Gal3 uptake in cells with perturbed clathrin function. Incubation of Gal3 and Tf for 15 min at 37°C with HeLa cells (a) or TECs (b) that expressed dominant negative GFP-tagged mutant of Eps15 (EH2-1). Cells were lactose and acid washed to remove plasma membrane accessible material. Tf endocytosis was strongly inhibited in mutant expressing HeLa cells and TECs, in contrast to Gal3. c,d, Gal3 uptake in cells with perturbed dynamin function. Incubation

of Gal3 and Tf for 15 min at 37°C with HeLa cells (c) or TECs (d) that expressed dominant negative GFP-tagged mutant dynamin (K44A). Cells were lactose and acid washed to remove plasma membrane accessible material. Gal3 endocytosis was not significantly perturbed in K44A expressing cells, while Tf endocytosis was strongly inhibited. Statistical analysis in this figure: Means ± S.D., n=3 independent experiments, 25-30 cells each. Student, unpaired t-test, * p<0.05, *** p<0.001. Scale bars = 10 µm.




a

b

Control

CTx

B-A

488

/ Gal

3-C

y3 PPMP

CTxBGal3

Surface fluorescence intensity(% of control)

On ice On ice

Gal3

Con

trol

Prot

eina

se K

Gal3 CTxBCTxB

On ice On ice On ice On ice

d

Gal

ectin

-4in

tera

ctio

n pa

rtner

s

Golgi


Plasma membrane

Unknown

Cytosol

Extracell. space

LysosomesMito.

Nucleus

Plasma membrane

Extracell. space

Cytosol Unknown

Lysosomes

Gal

ectin

-3in

tera

ctio

n pa

rtner

s

c Surface fluorescence intensity(% of control)

Control Tunicamycin Deoxymannojirimycin

CTx

B-A

488

/ Gal

3-C

y3

On ice On ice On ice

CTxB

10080604020

Gal3

Tunicamycin

Control

*120 140


***

***

PPMP

Control

10080604020 120

Supplementary Figure 4 Analysis of Gal3 uptake determinants. a, Gal3 surface binding on GSL-depleted cells. Control or PPMP-treated TECs were incubated on ice with Gal3 (red) and CTxB (green). On PPMP-treated cells, the binding of CTxB was strongly reduced, while Gal3 binding was not affected. Quantification of data on 12-15 cells per condition (means ± S.E.M; n=3 independent experiments). b, Protein determinants are required for Gal3 binding to cells. TECs were treated with proteinase K (right panel) or not (left panel) and incubated with Gal3 (red) and CTxB (green). Gal3 did not bind to proteinase K-treated cells, as opposed to CTxB. A representative result is

shown, n=3. c, Gal3 needs N-glycans for binding to cells. TECs were incubated or not for 24 hours with 6 µM tunicamycin or 48 hours with 90 μg*ml-1 1-deoxymannojirimycin, put on ice and incubated for 20 min with Gal3 (red) and CTxB (green). Gal3 binding was strongly impaired, but not that of CTxB. Quantification of data on 19-26 cells assessed from 4 fields per condition (means ± S.D., n=3 independent experiments). d, Sampling of Gal3 and Gal4 interacting partners according to intra and extracellular localizations. Protein identities are given in Supplementary Table 1. Statistical analysis in this figure: Students, unpaired t-test. *p<0.5, *** p<0.001. Scale bars = 10 µm.




a

Gal3 siRNA#1 Gal3 siRNA#2

PM

Scrambled siRNAPM

Untransfected

PM

g

Scrambled Gal3 Clathrin Gal3/Clathrin siRNA#3 siRNA#2 siRNA

Fluo

resc

ence

inte

nsity

(% o

f scr

ambl

ed)

**

40

80

60

20

100

120

e

Clathrin siRNA#2 Gal3/Clathrin siRNA


Scrambled

Gal3-siRNA

Control

b

Gal3

Tubulin

c

β1 integrin surface levels

Scrambled Gal3 Clathrin Gal3/Clathrin 20 nM 10 nM siRNA#3 siRNA#2 siRNA

Clathrin

Gal3

Actin

d

CD44 surface levels

40 8060 20 100 120


Scra

mbl

ed s

iRN

AG

al3

siR

NA

#2

CD44 Tf CD44 Tf Gal3

Gal

3 si

RN

A#1

Unt

rans

fect

ed

CD44, Tf-Alexa647 : 2 minCD44, Tf-Alexa647 : 2 min

Gal3-Cy3 : 30 min

PPM

PG

al3

siR

NA

#1

Scrambled Gal3

-

siRNA–

β1 integrin

Scrambled Gal3 siRNA#3

f

Supplementary Figure 5 CD44, b1-integrin, and fluid phase uptake in Gal3-depleted cells. a, Depletion of Gal3 on TECs using siRNA sequence #3. Western blot analysis in which tubulin served as a loading control. A representative result is shown. Corresponding cells were used for uptake experiments of Fig. 4d. Cells were tested for efficient knockdown in each experiment. b, CD44 cell surface levels on MEFs were not significantly altered upon Gal3 depletion. Untreated, scrambled siRNA transfected, or Gal3 depleted MEFs were incubated on ice with anti-CD44 antibodies, and after fixation with fluorescently labelled secondary antibodies. Means ± S.D., n=3 independent experiments, 15-35 cells assessed from 5 fields each. Corresponding cells were used for uptake experiments of Fig. 4d. c, Gal3 is required for CD44 uptake. Tf (blue) and anti-CD44 antibodies (green) were concomitantly incubated for the indicated times and in the indicated conditions with MEFs that were complemented or not with exogenous Gal3 (red). Cells were acid washed to remove plasma membrane accessible material. CD44 uptake was specifically reduced in Gal3-depleted MEFs, and exogenous Gal3 rescued this phenotype. Quantification is shown in Fig. 4c. d, Western blotting analysis of proteins expression in the indicated depletion

conditions. A representative result is shown. Corresponding cells were used for uptake experiments of Fig. 4g. Cells were tested for efficient knockdowns in each experiment. e, Examples of cells from the internalization analysis of Fig. 4g, using 9EG7 antibody against the active form of b1-integrin. f, b1-integrin cell surface levels in the indicated conditions, as determined by antibody binding on ice. Means ± S.D., n=3 independent experiments, 15-35 cells assessed from 5 fields each. Corresponds to experiments shown in Fig. 4g. g, EM analysis of HRP uptake in Gal3-depleted cells. Untransfected, scrambled siRNA, or Gal3 siRNA transfected MEFs were pulsed for 2 min with HRP (10 mg*ml-1) and processed for EM. In control conditions (untransfected or scrambled siRNA transfected cells), HRP-labeled tubular and ring-shaped structures were observed at early times of uptake (2 min, arrows). Some vesicular structures of different diameters were also labelled (arrowheads), likely representing clathrin-coated vesicles (80-120 nm) or detached caveolae (40-60 nm). On Gal3 siRNA transfected cells, the tubular and ring-shaped structures were strongly reduced, and mostly vesicular profiles were visualized. Quantification is shown in Fig. 4h. Statistical analysis: Student, unpaired t-test. ** p<0.01. Scale bars (c,e) = 10 µm, (g) = 200 nm.




d


g

Fluo

resc

ence

inte

nsity

(% o

f con

trol

)

40

80

60

20

100

**

LactoseControl Sucrose

FITC-Dextran uptake

e HRP-labeled carriers

Tubules, Rings (CLICs)Vesicles 80-120 nmVesicles40-60 nm

Sucr

ose

Lact

ose

Carriers / section - cell2015105

**

CLIC vs. Tf uptake

700

600

500

400

300

200

100

Fluo

resc

ence

inte

nsity

(% o

f suc

rose

2 m

in)

Tf-555

**

**

b

2 min 40 minSucroseLactose Sucrose Lactose

Anti-CD44

CD44 surface levels

SucroseLactose

Fluo

resc

ence

inte

nsity

(% o

f con

trol

)

Control

40

80

60

20

100

c

f

Control

5 m

in

Lactose

CD44 MergeTf-555

Suc

rose

Lac

tose

2 min

2 min

40 min

40 min

Suc

rose

Lac

tose

a

Sucrose

FITC-Dextran

Sucrose

Lactose

PM

PM

PM

PM

PM

PM

PM

PM

Supplementary Figure 6 CD44 uptake and fluid phase CLIC formation on cells incubated in the presence of lactose. a, Galectin function is required for CD44 uptake. MEFs were incubated with Tf (red) and anti-CD44 antibodies (green) for the indicated times in the presence of 100 mM sucrose or lactose. Cells were acid washed to remove plasma membrane accessible material. b, Labelling intensities of experiments as in (a) were quantified on 10 to 12 cells per condition (Means ± S.E.M., n=3 independent experiments) and normalized to the ‘Sucrose 2 min’ sample average for each marker, separately. Note that lactose specifically inhibited CD44 uptake. c, CD44 cell surface levels on MEFs were not significantly altered upon lactose incubation. MEFs in conditions as in (a) were incubated on ice with anti-CD44 antibodies, and after fixation with fluorescently labelled secondary antibodies. Means ± S.D. of 15 to 25 cells assessed from 5 fields per experiment, n=3 independent experiments. d, Fluid phase uptake is sensitive to galectin function. Lactose or sucrose-treated MEFs were pulsed for 2 min with HRP (10 mg*ml-1) and processed for EM. In control conditions (sucrose), HRP labelled tubular and

ring-shaped CLIC structures at early times of uptake (arrows). Some vesicular structures of different diameters were also labelled (arrowheads), likely representing clathrin-coated vesicles (80-120 nm) and detached caveolae (40-60 nm). In the presence of lactose, the occurrence of tubular and ring-shaped CLIC structures was largely reduced, and mostly vesicular profiles were visualized. e, Quantification of experiments as in (d) on 20-24 cells per condition (means ± S.E.M, n=3 independent experiments) confirmed a significant decrease of tubular and ring-shaped structures upon lactose treatment, while vesicles were not significantly affected. f, Fluid phase dextran uptake experiment. MEFs were incubated for 5 min with 1 mg*ml-1 of FITC-labelled dextran in the presence of the indicated sugars, fixed, and analysed for labelling intensity. Lactose significantly inhibited fluid phase endocytosis. g, Quantification of experiments as in (f). Means ± S.D. of 17 to 27 cells per experiment, n=3 independent experiments. Scale bars (a,f) = 10 µm, (d) = 200 nm. Statistical analysis in this figure: Student, unpaired t-test. ** p<0.01




Control

99% CI


Level of CD44 clustering

800600400200 1000

r (nm)KifunensineControl

Deoxymannojirimycin Swainsonine

CD44 ****


L(r)

-r a

t r =

50

nm

10050

250

150

0

200

Level of CD44 clusteringb

f

d


60

40

20

0

L(r)

— r

—20

HA-CD44 wt HA-CD44 5xN-Glyc mtHA-CD44

WT

Fluo

resc

ence

inte

nsity

(% o

f HA

-CD

44 w

t)

60

80

100

120

40

20

CD44 surface levels

HA-CD445xN-Glyc-mt

Surface levels

CD44 CD44

140

Control Kifunensine

Deoxymannojirimycin

Gal

3-A

488

/ CD

44 C

y3

Fluo

resc

ence

inte

nsity

(% o

f con

trol

)

80

20

40

60

Gal3-Alexa488CD44 Cy3120

100

Control Deoxy-manno-jirimycin

Kifunen-sine

Swain-sonine

**

Swainsonine

ca

e

g

Surface

Supplementary Figure 7 N-glycosylation in CD44 clustering and uptake. a, Clustering of Alexa-647-labeled anti-CD44 antibodies in control and 1-deoxymannojirimycin-treated MEFs. Mean Ripley’s K-function curves, L(r)-r, derived from single molecule localizations obtained with dSTORM imaging, plotted against radius, r, of concentric circles centred on each molecule relative to random distributions (99% confidence interval (CI) of simulated data). Averages of 15-20 cells, n=3 independent experiments. b, Maxima of Ripley’s K-function curves. Each symbol represents one image region; small horizontal lines indicate mean (± S.E.M., n=3 independent experiments). c, CD44 uptake in MEFs in which the biogenesis of complex type N-glycosylation

was perturbed or prevented with the indicated inhibitors. Cells were incubated for 2 min at 37°C with anti-CD44 antibodies. Quantification of uptake is shown in Fig. 5c. d,f, Cell surface levels of endocytic markers. MEFs in the indicated conditions were incubated on ice with anti-CD44 antibodies (revealed with appropriate fluorophore-labelled secondary antibodies) and Gal3-Alexa488 (d). e,g, Fluorescence was quantified after washing and fixation for data as in d and f, respectively. Means ± S.D., n=3 independent experiments with 21-28 cells assessed from 5 fields (e), n=4 independent experiments with 21-25 cells assessed from 7 fields (g). Statistical analysis in this figure: Student, unpaired t-test. ** p<0.01, **** p<0.0001.




25

35

5570

100130250

15Anti-IFNAR2

25

35

5570

100130250

15 Anti-beta1 integrin

25

35

5570

100130250

15Anti-actin

25

35

5570

100130250

15

Anti-CD44

Tubulin

Gal325

35

5570

100

130

250

15

Anti-Gal3

25355570

15

Clathrin

Actin2535

55

70100

130

250

15

70

kDa

kDa kDa

kDa

kDa

kDa

kDa


PPMPCtrl. GM95

MEB4CG1

CD44FITC

CD44FITC

25

35

5570

100130250

kDa

25

35

5570

100130

250

15

kDa

a

e

dc

b

i

gf

h

Supplementary Figure 8 Scans of uncropped blots. a-d, Corresponds to Fig. 3b. Western blotting analysis for Gal3 binding proteins. Blots were probed with anti-CD44 (a), anti-IFNAR2 (b), anti-b1 integrin (c) or anti-actin antibodies (d). e,f, Corresponds to Supplementary Fig. 1e. Biochemical CD44 uptake assay.

Cell lysates were analysed for anti-CD44-FITC by SDS-PAGE and fluorescence detection. g, Corresponds to Supplementary Fig. 5a. Cell lysates were probed against Gal3 and tubulin. h,i, Corresponds to Supplementary Fig. 5d. Cell lysates were probed against clathrin, actin (h) and Gal3 (i). kDa: Kilodalton.




Supplementary Table Legends

Supplementary Table 1: Listing of Gal3 and Gal4 interacting partners. Complete proteomics list of TEC proteins that were pulled down with Gal3 or Gal4. Hits were classified according to the number of peptides per protein bound to Gal3. Corresponds to Fig. 3a.

Supplementary Table 2: GSL expression in different cell lines. Thin layer chromatography (TLC), mass spectrometry (MS), and cholera toxin binding (biological assay, BA) were used to analyse GSL expression on TECs and MEFs. *isomeric molecules distinguished by TLC.

Supplementary Table 3: Listing of GSL species. Major molecular species of glycosphingolipids extracted from TEC and MEF cells were identified by shotgun mass spectrometry.

Supplementary Table 4: GUV summary table. Observed events within different GUV experiments are summarized. Corresponds to Fig. 7a-j.

Supplementary Table 5: Description of plasmids, primer and siRNAs used in this study.

Supplementary Video Legends

Supplementary Video 1: Gal3 is localized to CLICs in unperturbed MEFs. MEFs were incubated with Gal3-HRP for 2 minutes at 37°C, cooled on ice, labelled with DAB in the presence of ascorbic acid, fixed and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3-HRP positive carrier (green). Note the presence of invaginations in the Gal3-HRP carrier, as well as connected tubular extensions, similar to that previously described for CLICs. Image series was captured in IMOD and processed with ImageJ to 10 frames per second. Corresponds to Fig. 1e.

Supplementary Video 2: Gal3 is localized to CLICs in unperturbed TECs. TECs were incubated with Gal3-HRP for 2 minutes at 37°C, cooled on ice, labelled with DAB in the presence of ascorbic acid, fixed and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3-HRP positive carrier (green). Image series was captured in IMOD and processed with ImageJ to 10 frames per second.

Supplementary Video 3: Gal3 induces cisternal CLIC-like invaginations in ATP-depleted MEFs. ATP-depleted MEFs were incubated with Gal3-HRP and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3-HRP positive carriers (green and yellow), microtubules (orange) and actin microfilaments (blue). Complex, basket and ring shaped structures were observed with morphologies that were strikingly similar to previously captured tomography data of CLICs in unperturbed cells. Note the close proximity of actin and microtubules to Gal3-HRP positive invaginations. Image series was captured in IMOD and processed with ImageJ to 10 frames per second. Corresponds to Fig. 6c.

Supplementary Video 4: Gal3 induces tubular invaginations on GSL-containing GUVs, even in the presence of a-galactosidase. GUVs were prepared with a plasma membrane-like lipid composition containing Ni-lipids and a mix of GSLs, were treated with a-galactosidase for 30 min, subsequently incubated with 200 nM Gal3-His at 21°C, and imaged at the equatorial plane by confocal microscopy. Gal3-His induced invaginations after 30 min. Note: No invaginations formed on GUVs treated with b-galactosidase. Corresponds to Fig. 7f.


Supplementary Note 1:

dSTORM imaging and data processing

Sample preparation: Cells were incubated for 20 min at 37˚C with Gal3-Alexa647 at a

concentration of 4 µg*ml-1 in serum-free DMEM supplemented or not with PPMP,

and then fixed in 4% paraformaldehyde for 10 min at 37˚C. For Gal3-dependent

CD44 clustering in cells treated with 1-deoxymannojirimycin, cells were incubated

for 20 min at 37˚C with Gal3 at a concentration of 4 µg*ml-1 in the presence of 1-

deoxymannojirimycin. Subsequently, cells were fixed in 4% paraformaldehyde for 10

min at 37˚C and stained for CD44 using an Alexa-647 labelled antibodies directed

against CD44.

Reversible photoswitching of Alexa-647 fluorophores in dSTORM

experiments was performed in a redox buffer composed of 25 mM Hepes and 5%

glycerol in PBS, pH 8, using an oxygen scavenger system made of 0.05 mg*ml-1

glucose oxidase, 0.025 mg*ml-1 horseradish peroxidase, 75 mM cysteamine, and 25

mM glucose1. dSTORM images were acquired on a TIRF microscope (ELYRA;

Zeiss) with a 100x, NA=1.46 oil-immersion objective. For imaging, a 633 nm laser

was used for excitation and a 488 nm laser for conversion from the dark state. 15,000-

20,000 images were acquired per sample using an electron-multiplying CCD

(EMCCD) camera (iXon DU-897D, Andor) with an exposure time of 40 ms.

For data processing, point spread function (PSF) fitting and localization was

performed in Zen 2010D (Zeiss Microscopy). Single molecules were identified as

areas with an intensity-to-noise ratio greater than 6. The centre of each PSF was

calculated by fitting to a 2-dimensional Gaussian distribution. Sample drift was

corrected by tracking immobile gold fiducial markers. To prevent over-counting due


to fluorophore blinking, localizations that were dark for less than 1,200 ms before re-

exciting were grouped into a single localization.

From each dSTORM image, 4 µm x 4 µm non-overlapping regions were

selected for analysis. Events with localization precision worse than 50 nm were

discarded. For each analysed region, Ripley’s K-function was calculated as:

K r = A (!!"!!

!!!!

!!!! ) where δ!" = 1 if δ!" < r, else 0 Equation 1

Here, n is the number of points contained in an analysed region of area A (4 µm x 4

µm). dij is the distance between two points i and j and r is the analysed spatial scale.

Localizations at the edge of the analysed region were weighted to negate edge effects.

The K-function is a measure of the number of points encircled within concentric

circles of radius r centred on each point and scales with circle area. It is therefore

transformed into the L-function to obtain linear scaling with radius, r via Equation 2:

! ! = !(!)!

Equation 2

Random distributions have L(r)=r at all r. We therefore plot L(r)-r versus r. Positive

values of L(r)-r at a given r indicate clustering at that spatial scale. 99% confidence

intervals (CI) were generated by simulating 100 spatially random distributions.

To generate cluster heat maps, L(r) values at a spatial scale of 50 nm, L(50),

were computed for each point individually (Equation 3).

!! 50 =! (

!!"!

!!!! )

! where δ!" = 1 !" δ!" < 50, !"#! 0 Equation 3


Values of L(50) were interpolated to form a cluster heat map (MATLAB, The

Mathworks). By applying a threshold of L(50)=78 to this map, a binary cluster map

was created from which cluster statistics (number, size, shape, molecules/cluster) was

extracted. This method has previously been demonstrated to extract clustering

parameters from PALM and dSTORM data at the plasma membrane1-3. Statistical

significance of the means of two data sets was assessed with Student t-tests (Prism,

GraphPad Software).

Crucial to PALM and dSTORM data analysis is the correct identification of

single molecules and avoidance of over-counting molecules whose fluorescence has

ceased and re-appeared. To correct for this effect we used an accepted method of

analysis first published by Annibale et al.4, and later modified by Rossy et al.1 to

include two different time-scales of blinking. We fitted Gal3-Alexa647 data to the

following equation (4):

! t! = N 1+ !!"#$%!!( !!!!!!""!

) + !!"#$%!!

( !!!!!!""!)

Equation 4

Here N(td) is the number of molecular counts at different off gaps (td) where off-gap

is the maximum time period a molecule is dark before being identified as a different

molecule when its fluorescence resumes; N is the number of fluorescent molecules;

nblink1 and nblink2 are the average number of counts a molecule converts to a dark

state; toff1 and toff2 is the average lifetime of the fluorophore. We corrected for the

factors described above using clustering data of HeLa cells treated with PPMP and

untreated control cells. As an additional control, we have performed dSTORM

experiments with Gal3-Alexa647 molecules adhered to clean coverslips imaged under


the identical parameters as the cell samples. For all samples the duration of the

acquisition was 20,000 frames at 30 ms per frame. We plotted the number of localized

Gal3-Alexa647 molecules in representative images versus the off-gap and fitted the

data to equation 4 (Supplementary Figure 2i-k).

Alexa647-Gal3 exhibited 0.25 blinks for 0.26 frames (7.8 ms) and 0.77

blinks for 6.2 frames (186 ms) in HeLa control cells, and 0.16 blinks for 0.25 frames

(7.5 ms) and 0.44 blinks for 7.7 frames (231 ms) in PPMP treated cells. Alexa647-

Gal3 molecules adhered to coverslips displayed 0.19 blinks per molecule for 0.5

frames (15 ms) and 0.2 blinks for 22 frames (660 ms). Based on this analysis, an off-

gap threshold of 40 frames was applied.

With this threshold, and the exclusion of molecules with localisation

precisions >50 nm, Alexa647-Gal3 molecules adhered to clean coverslips was not

completely random (probably due to Gal3 aggregation in solution and aggregation of

denatured Gal3 on the surface), but was 6-fold lower than Galectin3-Alexa647

molecules in HeLa (Supplementary Figure 2h).

1. Rossy, J., Owen, D.M., Williamson, D., Yang, Z. & Gaus, K. Conformational states of Lck regulate clustering in early T cell signalling. Nat. Immunol. 14, 82-‐89 (2013).

2. Owen, D.M. et al. PALM imaging and cluster analysis of protein heterogeneity at the cell surface. J. Biophotonics 3, 446-‐454 (2010).

3. Williamson, D.J. et al. Pre-‐existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12, 655-‐662 (2011).

4. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Quantitative photo activated localization microscopy: unraveling the effects of photoblinking. PLoS ONE 6, e22678 (2011).


galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

Documents