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Myosin 1b promotes the formation of post-Golgicarriers by regulating actin assembly and membraneremodelling at the trans-Golgi networkClaudia G. Almeida1,2, Ayako Yamada1,3, Danièle Tenza1,4,5, Daniel Louvard1,2, Graça Raposo1,4,5

and Evelyne Coudrier1,2,6

The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, thecytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have beenimplicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute tothe membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1bregulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, togetherwith the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation byregulating actin assembly and remodelling TGN membranes.

The trans-Golgi network (TGN) is one of the main proteinsorting stations of the cell at the crossroads of the exocytic andendocytic pathways. This organelle is composed of a complex tubularnetwork that emanates from the trans-Golgi cisternae, and generatespleiomorphic carriers targeted to different destinations. Its shapeis intimately associated with its function. Marked changes in itsmembrane shape underlie the exit of cargos. This exit involvesconcentration of cargo in membrane domains of the TGN, membranedeformation or budding, elongation of tubular-carrier precursorsand scission leading to the formation of the post-Golgi carriers.Post-Golgi carriers are then transported through the cytosol alongmicrotubules. There are various possible mechanisms that canregulate membrane deformation, which include changes in lipidcomposition, oligomerization of scaffolding proteins or insertion oftransmembrane proteins, that through their shape or clustering canbend membranes1. The cytoskeleton and its motors, which can changethe mechanical properties of membranes, also play a major role inmembrane deformation1.Growing evidence indicates that the actin cytoskeleton plays a

role in membrane trafficking at the TGN, and numerous proteinsrelated to the actin-based system have been now localized to thisorganelle2–5. However the role of actin dynamics at an early stage ofpost-Golgi carrier biogenesis, such as membrane deformation, is much

1Institut Curie, Centre de Recherche, Paris, F-75248, France. 2Morphogenesis and Cell Signalization CNRS, UMR144, Paris, F-75248, France. 3Cell and TissueImaging Facility, CNRS UMR 144, Paris F-75248, France. 4Membrane and Cell Functions, CNRS UMR 168, Paris F-75248, France. 5Structure and MembraneCompartments CNRS, UMR144, Paris, F-75248, France.6Correspondence should be addressed to E.C. (e-mail: evelyne.coudrier@curie.fr)

Received 3 June 2010; accepted 18 April 2011; published online 12 June 2011; DOI: 10.1038/ncb2262

less documented. Two myosins, myosin 6 and myosin 2, contributeto post-Golgi traffic6–8 and recent evidence indicates that myosin 2 isrequired for scission9, but the possible role of amyosin in an earlier stepof the post-Golgi carrier biogenesis remains to be established. In thisreport we show that a myosin 1 (myosin 1b) and the actin cytoskeletonpromote the formation of tubular-carrier precursors. Together, ourdata demonstrate that myosin 1b is a key regulator of membraneremodelling at the TGN by controlling filamentous actin (F-actin)assembly in this region.

RESULTSA pool of myosin 1b is associated with F-actin foci andCI-mannose-6-phosphate receptor in the perinuclear regionMyosin 1b (Myo1b) is involved in the traffic of cargo along theendocytic pathway10–12. It has been localized at the plasma membranein regions enriched for actin filaments and at early endosomes,multivesicular endosomes and lysosomes11–13. A pool of Myo1bwas also found to co-fractionate with Golgi markers and detectedby immunofluorescence in the perinuclear region of mouse hep-atoma cells11,14. In agreement with these observations, we observedan accumulation of Myo1b in the perinuclear region of HeLacells (Fig. 1a). This pool co-localizes partially with TGN46 andCI-mannose-6-phophate receptor (MPR) that carries cargos from

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Figure 1 Distribution of Myo1b, MPR, F-actin and the Arp2/3 complexin the perinuclear region. (a) A representative spinning-disc confocalmicroscopy section throughout the nucleus after detection of Myo1bby immunofluorescence in HeLa cells. Note the concentration ofMyo1b in the perinuclear region. The scale bar represents 10 µm. (b) Arepresentative spinning-disc confocal microscopy section throughout thenucleus after detection of Myo1b (green), TGN46 (blue) and MPR (red)by immunofluorescence. Note the overlapping distribution of Myo1b withMPR and TGN46 (arrows). The white box indicates the enlarged TGNregion. The scale bar represents 10 µm. (c) HeLa cells co-transfectedwith plasmids encoding GFP–Myo1b and LifeAct–Cherry were analysedby spinning-disc confocal microscopy. A representative spinning-disc

confocal microscopy section through the nucleus shows the overlappingdistribution of GFP–Myo1b (green) and F-actin foci detected withLifeAct–Cherry (red). The white box indicates the enlarged TGN region.Note the overlapping distribution of Myo1b with F-actin foci in theTGN region (arrows). The scale bar represents 10 µm. (d) HeLa cellsco-transfected with plasmids encoding GFP–Myo1b and p16–Cherry, asubunit of the Arp2/3 complex, were analysed by spinning-disc confocalmicroscopy. A representative spinning-disc confocal microscopy sectionthrough the nucleus shows the overlapping distribution of GFP–Myo1b(green) with p16–Cherry (red). The white box indicates the enlargedTGN region. Note the overlapping distribution of Myo1b with the Arp2/3complex in the TGN region (arrows). The scale bar represents 10 µm.

the TGN to sorting endosomes and recycles back to TGN (ref. 15;Fig. 1b).Because myosins function with F-actin we looked for these polymers

in the TGN region. As previously reported, we observed short actin fil-aments also named F-actin foci in a focal plane above actin stress fibresin live HeLa cells transfected with the F-actin-binding peptide Life-Act–Cherry (Fig. 1c; refs 3,4,11,16). We also detected F-actin foci onfixed cells by using fluorescent phalloidin that codistributed with a sub-unit of the Arp2/3 complex (p34) promoting actin assembly by nucleat-ing branched actin filaments (Supplementary Fig. S1). A subpopulationof green fluorescent protein (GFP)–Myo1b overlappedwith F-actin fociand with a subunit of the Arp2/3 complex (p16) in live cells (Fig. 1c,d;arrows). We concluded that a pool of Myo1b is often in close proximitywith F-actin foci andMPR-positivemembranes in the TGN region.

Myo1b expression regulates MPR distributionThe proximity of Myo1b to MPR-positive membranes and F-actinfoci in the perinuclear region prompted us to investigate whetherMyo1b participates in the traffic of cargo at the TGN. We analysed

the cellular distribution of MPR on knockdown or overexpression ofMyo1b. Myo1b was hardly detectable by immunofluorescence andWestern blot in HeLa cells transfected with a home-designed shortinterfering RNA (Myo1bH siRNA) as well as two siRNAs designed byDharmacon (Myo1b-D3 and D4 siRNAs; Supplementary Fig. S2a,b).Yet the reduction observed in cells transfected withMyo1bH siRNAwas slightly higher (up to 95% reduction) when compared with cellstransfected withMyo1b-D3 siRNA andMyo1b-D4 siRNA (up to 69%and 94% respectively; Supplementary Fig. S2c). Thus we have pursuedthe majority of our study withMyo1bH siRNA.Knockdown of Myo1b by transfecting HeLa cells with the

three Myo1b siRNAs altered the distribution of MPR (Fig. 2a andSupplementary Fig. S2d). Cells transfected with control siRNA showeda perinuclear staining that codistributed with TGN46 and a prominentpunctuate staining in the cytoplasm consistent with the endosomalpool of MPR (Supplementary Fig. S2d). Indeed, this second poolcodistributed partially with sorting endosomes identified by sortingnexin 1 (SNX1) (ref. 17; Supplementary Fig. S2f). In contrast, MPRwas almost exclusively localized in the TGN region of cells depleted

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(c) HeLa cells were transfected with plasmids encoding GFP (green)or GFP–Myo1b (green) and immunolabelled with anti-MPR antibody(red), and the distribution of MPR was analysed by confocal microscopy.Representative maximum projections are shown. The scale bars represent10 µm. (d) The number of cells exhibiting a dispersed distribution of MPRas shown for GFP–Myo1b-expressing cells or a normal distribution asshown for GFP-expressing cells was counted and normalized to the totalnumber of cells analysed in three independent experiments (n=3,N =97GFP–Myo1b-expressing cells, N =187 GFP-expressing cells).

for Myo1b (Fig. 2a and Supplementary Fig. S2d). More than 90%of the immunofluorescence labelling corresponding to MPR wasdetected in the perinuclear region of cells treated with the threeMyo1bsiRNAs, compared with 60% in control-siRNA-treated cells (Fig. 2band Supplementary Fig. S2e).In contrast to cells knocked down for Myo1b, 44% of cells

overexpressing GFP–Myo1b as compared with 17% of the GFP-expressing cells did not show an accumulation of MPR in the TGNregion (Fig. 2c,d). This behaviour is probably due to a scattered TGNand MPR associated with SNX1-labelled endosomes (SupplementaryFig. S2f,g). Together these observations led us to postulate that Myo1bregulates the traffic of MPR.

Myo1b modulates TGN exit of MPR and p75 but notGPI-anchored proteinsWe previously reported that expression of dominant negative mutantsor overexpression of Myo1b altered traffic along the endocytic pathway.To address the functional importance of Myo1b in post-Golgi trafficwe thus investigated first whether Myo1b knockdown altered the trafficof MPR from endosomes to the TGN. We found that endocytosedMPR reached the TGN region at the same rate in control- andMyo1b-siRNA-treated cells (Fig. 3a,b). Similarly, the traffic of theGb3-binding B-subunit of Shiga toxin (STxB) from endosomes toTGN was not altered in Myo1b knockdown cells (Supplementary Fig.S3a,b). Using cryo-electron microscopy we observed that STxB wastransported to the TGN (Supplementary Fig. S3c).We next determined whether Myo1b depletion could affect the

MPR exit from the TGN. The exit of the GFP–MPR from the TGNwas monitored after a temperature block at 20 ◦C that inducedits accumulation in the Golgi complex of control-siRNA- andMyo1b-siRNA-transfected cells (Fig. 3c and Supplementary Movie S1;refs 18,19). The fluorescence intensity of GFP–MPR decreased in

the perinuclear region on the release of the temperature block incontrol-siRNA-transfected cells. After 70min, the distribution of MPRbetween the TGN and endosomes reached its equilibrium. The amountof MPR in the TGN decreased by 30% of its initial fluorescence incontrol-siRNA-transfected cells. In contrast, the amount of MPRdecreased only by 15% of the initial fluorescence within 80min inMyo1b-siRNA-treated cells (Fig. 3d).As a significant amount of MPR is retained in the TGN on Myo1b

knockdown the delivery of newly synthesized lysosomal hydrolases,such as cathepsin D, to endosomes should be affected. Indeed, thefluorescence corresponding to cathepsin D increased by 40% inthe TGN region of Myo1b-siRNA-treated cells as compared withcontrol-siRNA-treated cells (Fig. 3e,f); its processing was slightlydelayed (Supplementary Fig. S4a,b) and it co-localized less with dextran-labelled compartments (Supplementary Fig. S4c). By contrast, thecellular distribution of LAMP1, a marker of late endosomes/lysosomes,was not altered onMyo1b depletion (Supplementary Fig. S4d,e).If the delivery of lysosomal hydrolases to lysosomes is impaired on

Myo1b knockdown they may instead be secreted as previously reportedin cells knocked out for MPR (ref. 20). Surprisingly, we found adecrease of nearly 40% for the activity ofβ-hexosaminidase, one of theselysosomal hydrolases, in cell-culture media ofMyo1b-siRNA-treatedcells as compared with control-siRNA-treated cells (Fig. 3g). Onepossible interpretation of this observation is that depletion of Myo1binhibits not only MPR post-Golgi transport to endosomes butalso post-Golgi transport to the plasma membrane. In agreementwith this hypothesis the kinetics of the exit from the TGN of theneurotrophin receptor p75 that localizes at the plasma membrane atsteady state19 was slowed down inMyo1b-siRNA-treated cells. After120min, the amount of p75 left in the TGN was less than 10% incontrol-siRNA-treated cells, whereas in Myo1b-siRNA-treated cellsit remained more than 40% (Fig. 3h and Supplementary Movie S2).

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Figure 3 Myo1b knockdown impairs the TGN exit of cargo. (a,b) Theinternalized MPR in the perinuclear region was analysed by epifluorescencemicroscopy (a) and quantified (b). HeLa cells were transfectedwith control siRNA or Myo1b siRNA and with a plasmid encodingchMPR (bovine murine MPR chimera). Cells were then labelled withanti-chMPR antibody for 5min followed immediately by fixation (0min),or incubated for 60min and then labelled with antibody followed byfixation (60min). The scale bar represents 10 µm (n = 2,N = 8–10control-siRNA-treated cells, N =7–12 Myo1b-siRNA-treated cells, mean).(c) The kinetics of the exit of GFP–MPR from the TGN was monitoredby time-lapse imaging using spinning-disc confocal microscopy incontrol-siRNA- and Myo1b-siRNA-treated HeLa cells after temperatureblock (see Supplementary Movie S1). Representative maximum intensityprojections at 0 and 120min are shown. The scale bar represents 10 µm.(d) The amount of GFP–MPR fluorescence was quantified in the TGNregion as a function of time and as a percentage of the fluorescencedetected at 0min (n = 3,N = 16 control-siRNA-treated cells, N = 21Myo1b-siRNA-treated cells; mean±s.e.m.). (e) HeLa cells were transfectedwith Myo1b siRNA or control siRNA, immunolabelled with anti-cathepsinD and TGN46 antibodies and analysed by epifluorescence microscopy.

The scale bar represents 10 µm. (f) The amount of cathepsin D in theTGN region was quantified (n = 3,N = 47 control-siRNA-treated cells,N = 67 Myo1b-siRNA-treated cells; ∗P = 10−14, t -test, mean± s.e.m.).(g) β-hexosaminidase activity was quantified in conditioned media ofcontrol-siRNA- and Myo1b-siRNA-treated HeLa cells (n = 2, carriedout in duplicate; mean). (h) The kinetics of exit of p75–GFP fromthe TGN (see Supplementary Movie S2) was analysed as describedin c,d for GFP–MPR exit (n = 4,N = 77 control-siRNA-treated cells,N =58 Myo1b-siRNA-treated cells; mean±s.e.m.). (i) Arrival of p75–GFPat the plasma membrane in control-siRNA- and Myo1b-siRNA-treatedHeLa cells was quantified as a function of time (n = 3, N = 47–61control-siRNA-treated cells, N =38–75 cells Myo1b-siRNA-treated cells;mean± s.e.m.). (j) The kinetics of exit of GPI–GFP from the TGN(see Supplementary Movie S3) was analysed as described in c,d forGFP–MPR (n = 3,N = 72 control-siRNA-treated cells, N = 68 cellsMyo1b-siRNA-treated cells; mean±s.e.m.). (k) HeLa cells transfected withcontrol siRNA or Myo1b siRNA were analysed by conventional electronmicroscopy. On Myo1b depletion, Golgi stacks are more compact (lowmagnification; left panel), shorter and often more dilated at the rims (highmagnification, arrows; right panels). The scale bars represent 250nm.

Furthermore, we observed that the delivery of p75–GFP to the plasmamembrane was similarly inhibited by 40% on Myo1b knockdown(Fig. 3i and Supplementary Fig. S4f).

Together, these observations led us to conclude that Myo1b at theTGN controls the traffic of MPR to endosomes, the exocytosis ofβ-hexosaminidase and the traffic of p75 to the plasma membrane.

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Interestingly, the exit of MPR and p75 depends on an intact actincytoskeleton5,18,21. In contrast, the exit of a lipid-raft-anchoredprotein, glycosyl phosphatidylinositol (GPI)–GFP, reported to exitthe TGN independently of the actin cytoskeleton5,21, was similarin control-siRNA- and Myo1b-siRNA-treated cells (Fig. 3j andSupplementary Movie S3).

Myo1b contributes to Golgi and TGN ultrastructurePrevious reports have shown that knockdown of proteins that impedepost-Golgi traffic perturbs Golgi and TGN morphology3,4,22. As thelack of Myo1b inhibits cargo exit and induces their accumulationin the TGN, we addressed whether it could subsequently leadto morphological alteration of the Golgi complex and the TGN.Depletion of Myo1b produced shorter Golgi stacks, dilated mostlyat the rims, and an abnormal accumulation of peri-Golgi vesicularprofiles with various shapes and sizes when analysed by conventionalelectron microscopy (Fig. 3k). A plausible interpretation of theseobservations is that inhibition of TGN cargo exit on Myo1b depletionimpairs the homeostasis of the Golgi complex and consequentlydisturbs its morphology.

Myo1b regulates MPR post-Golgi carrier tubule formationExit of MPR from the TGN depends on the formation of tubular-carrier precursors that elongate and break into post-Golgi carriers2,23.We thus investigated whether knockdown or overexpression ofMyo1b alters the formation of post-Golgi carriers loaded withGFP–MPR by fast time-lapse imaging. Consistent with previousobservations, we observed the formation of dynamic tubules containingGFP–MPR that extended from the TGN as well as numerouscarriers moving in the cytoplasm of control-siRNA-transfected cells(Fig. 4a and Supplementary Movie S4). Occasionally, F-actin fociand Myo1b were recruited to the TGN before the elongationof a tubular precursor (Fig. 4d,e and Supplementary Movie S5).In striking contrast to control-siRNA-treated cells, Myo1b-siRNA-treated cells showed a marked reduction in cytoplasmic carriers(Fig. 4a and Supplementary Movie S4). The number of GFP–MPRcarriers was reduced by 70% in Myo1b-siRNA-treated cells whencompared with control-siRNA-treated cells (Fig. 4b). This is probablya consequence of a decrease by 65% of the formation of tubularprecursors inMyo1b-siRNA-treated cells when compared with control-siRNA-treated cells (Fig. 4c). In contrast, the overexpression ofCherry–Myo1b increased by 2.5-fold the number of long GFP–MPRtubular precursors as compared with mock cells without affectingthe number of cytoplasmic carriers (Fig. 4f–h and SupplementaryMovie S6). Indeed tubular precursors were rather stable, positivefor TGN46, and no scission was observed during the 1min movie,indicating that scission is the limiting step of carrier formation inthese experimental conditions (Supplementary Fig. S5 and Movie S6).We concluded that Myo1b is required for the formation of tubular-precursor carriers.

Myo1b regulates F-actin fociMyo1b belongs to the short-tail myosin 1 subgroup. This subgroupdoes not contain a second actin-binding domain or a protein–proteininteracting motif such as a single Src homology domain 3 (SH3)that can bind proteins involved in actin polymerization24. However,another member of the short-tail myosin 1 subgroup, myosin 1c,

has been suggested to spatially control actin assembly to the plasmamembrane in Xenopus oocytes25. Similarly, Myo1b may couple actinassembly to the membrane of the TGN. To test this hypothesiswe monitored the behaviour of F-actin foci at the TGN in cellsexpressing Cherry–LifeAct and GFP–MPR on depletion of Myo1b.The F-actin foci remained relatively non-motile, and did not form ordisappear during the time of the movies in control-siRNA-treated cells(Fig. 5a,b and Supplementary Movie S7). 30% of them codistributedwith the MPR TGN pool (Fig. 5d). The number of F-actin foci wasreduced by 59% and the number of overlapping puncta betweenthe F-actin foci and MPR membranes in the TGN region wasdecreased by 59% in Myo1b-siRNA-treated cells as compared withcontrol-siRNA-treated cells (Fig. 5c,d and Supplementary Movie S7).These observations indicate that the interaction between F-actinfoci and MPR membranes decreased on Myo1b depletion. Thenumber of F-actin foci when detected with phalloidin on fixedcells was decreased in similar proportion (62%) in Myo1b-siRNA-treated cells as compared with control-siRNA-treated cells whereasthe number of stress fibres at the base of the cells was similarin control siRNA- and Myo1b-siRNA-treated cells (Fig. 5e,f andSupplementary S6a,b).To confirm that F-actin foci in the perinuclear region decreased

on depletion of Myo1b, we analysed the distribution of the Arp2/3complex that co-distributes with F-actin foci using antibodies againstthe p34 subunit (Fig. 6c). The number of Arp2/3 structures in thevolume occupied by MPR in the TGN area decreased by 54% whereasit decreased by only 12% at the ventral plasma membrane on depletionof Myo1b (Fig. 6d,e).To further confirm that Myo1b recruits F-actin foci in the

TGN region we analysed the impact of Myo1b overexpression onthe distribution of F-actin and the Arp2/3 complex. 79% of thecells expressing Cherry–Myo1b, compared with 15% of mock cells,presented fewer actin stress fibres (Supplementary Fig. S6c,d). Thesecells also presented an increased number of Arp2/3-labelled structuresin the cytoplasm (Fig. 5g). We quantified the number of Arp2/3structures in the volume occupied by GM130, a protein associated withthe cis-Golgi. Arp2/3 structures were increased more than eightfold inthis region whereas no significant increase was observed at the ventralplasma membrane (Fig. 5h,i). Taken together, these observationsindicate thatMyo1b tethers F-actin foci to TGNmembranes.

Motor activity of Myo1b is required to maintain MPR and F-actinfoci distribution at the TGNWe next determine whether the force generated by Myo1b is requiredto control the distribution of MPR and F-actin foci at the TGN.We first rescued the steady-state distribution of MPR as well asthe distribution of F-actin foci by expressing low levels of Flaghaemagglutinin (FlagHA)–Myo1b-5M (Fig. 6a,b). These observationsconfirmed the specificity of Myo1b siRNA. We used our ability torescue Myo1b knockdown with FlagHA–Myo1b-5M to determinewhether the motor activity of Myo1b is required to control thedistribution of MPR and F-actin foci. We designed a Myo1b rigormutant by introducing the mutation N160A in the ATPase pocket ofFlagHA–Myo1b-5M (FlagHA–Myo1b-5MR) and a second mutant byintroducing the mutation E409K in ‘switch 2’ of the motor domain(FlagHA–Myo1b-5ME) corresponding to Dictyostellium myosin 2

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Figure 4 Myo1b controls the formation of tubular carriers at the TGN.(a) HeLa cells were transfected with Myo1b siRNA or control siRNA, andwith a plasmid encoding GFP–MPR. GFP–MPR carriers were monitoredat 37 ◦C by time-lapse imaging using spinning-disc confocal microscopy(see Supplementary Movie S4). The first frames of representative moviesand representative kymographs (that cover 26 s) revealing the sequenceof events of tubule formation39 and scission (∗) are shown. The arrowindicates the beginning of tubule formation. The scale bars represent10 µm. (b) The average number of cytoplasmic carriers observed perframe was quantified (n = 3,N = 24 control-siRNA-treated cells, N = 19Myo1b-siRNA-treated cells; ∗P = 10−5, t -test, mean± s.e.m.). (c) Thenumber of tubules that formed and underwent scission per minute wasquantified (n = 3,N = 247 tubules in 21 control-siRNA-treated cells,N = 103 tubules in 25 Myo1b-siRNA-treated cells; ∗P = 10−7 t -test,mean±s.e.m.). (d,e) HeLa cells were co-transfected with plasmids encodingGFP–MPR and LifeAct–Cherry (d) or GFP–MPR and Cherry–Myo1b (e).The first frames of representative movies and representative kymographsof cells expressing low levels of the recombinant proteins (that cover 6 s)

revealed the presence of Cherry–Myo1b (red; see Supplementary Movie S5)and LifeAct–Cherry (red) at the base of nascent GFP–MPR (green) tubulesemanating from the TGN (arrows; ref. 39). The scale bars represent 10 µm.A schematic representation of the distribution of Cherry–Myo1b (red) andLifeAct–Cherry (red) at the base of the nascent tubes (green) is also shown.(f) HeLa cells were co-transfected with plasmids encoding GFP–MPR (green)and Cherry–Myo1b (red) or with GFP–MPR alone (Mock). GFP–MPR carrierswere monitored at 37 ◦C by time-lapse imaging using spinning-disc confocalmicroscopy (see Supplementary Movie S6). The first frames of representativemovies and representative kymographs (that cover 26 s) revealing thesequence of tubule formation39 and scission (∗) are shown. Note that thetubule did not undergo scission in the Cherry–Myo1b-expressing cell. Thearrow indicates the beginning of tubule formation. The scale bar represents10 µm. (g) The average number of cytoplasmic carriers observed per framewas quantified (n = 3, N = 28 Cherry–Myo1b-expressing cells, N = 24mock cells; P =0.87, t -test mean±s.e.m.). (h) The number of GFP–MPRstable tubules was quantified (n=4,N =49 Cherry–Myo1b-expressing cells,N =40 mock cells; ∗P =0.0014 t -test, mean±s.e.m.).

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are shown at low magnification. The white boxes indicate the enlargedGolgi regions. A single focal plane of F-actin in the Golgi region is shownat high magnification. The scale bar represents 10 µm. (f) The number ofF-actin foci in the Golgi region was quantified as described in the Methods(n =2,N =12 control-siRNA-treated cells, N =18 Myo1b-siRNA-treatedcells). (g) HeLa cells were transfected with plasmids encoding Cherry orCherry–Myo1b, immunolabelled with anti-p34 and GM130 antibodies andanalysed by three-dimensional deconvolution microscopy. Representativemaximum-intensity projections of Cherry (red), Cherry–Myo1b (red) andmerged p34 (green) and GM130 (red) are shown at low magnification. Thewhite boxes indicate the enlarged TGN region. Single focal planes of p34 inthe Golgi region are shown at high magnification. The scale bar represents10 µm. Note the increase of p34 puncta in Cherry–Myo1b-expressingcells in the Golgi region. (h,i) The number of p34 puncta in the volumeoccupied by GM130 in the Golgi region (∗P = 0.0032, t -test, h) andat the ventral plasma membrane (P = 0.26, t -test, i) was quantified asdescribed in the Methods (n =3,N =13 Cherry-expressing cells, N =21Cherry–Myo1b-expressing cells).

N233A and E476K mutants respectively26,27. The N233A rigormutants remain bound to F-actin whereas the E476K mutants showa weak affinity for F-actin. Similarly to Dictyostellium myosin 2,FlagHA–Myo1b-5MR and FlagHA–Myo1b-5ME were unable to

generate force tomove F-actin in vitro and FlagHA–Myo1b-5MEboundweakly to F-actin (Supplementary Fig. S7a). FlagHA–Myo1b-5MRdistributed in the perinuclear region whereas FlagHA–Myo1b-5MEappeared to be cytosolic, indicating that the E409K mutation alters the

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Figure 6 Functional Myo1b is required for organizing F-actin–Arp2/3foci. (a) HeLa cells were transfected with control siRNA orMyo1b siRNA and with plasmids encoding FlagHA–Myo1b-5M,FlagHA–Myo1b-5MR or FlagHA–Myo1b-5ME, immunolabelled withanti-MPR (green), anti-TGN46 (red) and anti-HA antibodies andanalysed by epifluorescence microscopy. The scale bar represents10 µm. (b) The percentage of fluorescence corresponding to MPRin the TGN region was quantified as described in the Methods(n =3,N =55 control-siRNA-treated cells, N =54 Myo1b-siRNA-treatedcells, N = 41 Myo1b siRNA + FlagHA–Myo1b-5M-treated cells,N = 46 Myo1b siRNA + FlagHA–Myo1b-5MR treated cells, N = 56Myo1b siRNA + FlagHA–Myo1b-5ME treated cells, ∗P < 0.05 versuscontrol-siRNA-treated cells, analysis of variance (ANOVA), mean±s.e.m.).(c) HeLa cells were transfected with control siRNA or Myo1b siRNAand with plasmids encoding FlagHA–Myo1b-5M, FlagHA–Myo1b-5MR

or FlagHA–Myo1b-5ME, immunolabelled with anti-p34 (green), HA andMPR (red) antibodies and analysed by three-dimensional deconvolutionmicroscopy. Representative maximum-intensity projections at lowmagnification of HA and merged MPR (red) and p34 (green) are shown.Single focal planes at high magnification of merged MPR and p34,and p34 alone, are shown. The white boxes indicate the enlargedTGN region. The scale bar represents 10 µm. (d,e) The number of p34puncta in the volume occupied by MPR in the TGN region (∗P < 0.05,versus control-siRNA-treated cells, ANOVA, mean±s.e.m., d) and at theventral plasma membrane (P > 0.05, versus control-siRNA-treated cells,ANOVA, mean± s.e.m., e) was quantified as described in the Methods(n =3,N =18 control-siRNA-treated cells, N =17 Myo1b-siRNA-treatedcells, N = 22 Myo1b siRNA + FlagHA–Myo1b-5M-treated cells, N = 22Myo1b siRNA + FlagHA–Myo1b-5MR treated cells, N =19 Myo1b siRNA+ FlagHA–Myo1b-5ME treated cells).

ability of Myo1b to bind to membranes (Fig. 6a and SupplementaryFig. S7b). FlagHA–Myo1b-5MR as well as FlagHA–Myo1b-5ME failedto rescue the normal steady-state distributions of MPR and F-actin foci

(Fig. 6). We concluded that the force generated by Myo1b is requiredto maintain the steady-state distribution of MPR and to tether or toorganize the F-actin foci at the TGN.

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Figure 7 Depletion of the Arp2/3 complex induces MPR accumulationin TGN and inhibits post-Golgi carrier formation. (a) HeLa cells weretransfected with p34-A siRNA or control siRNA, immunolabelled withanti-p34 and GM130 antibodies, labelled with fluorescent phalloidin andanalysed by three-dimensional deconvolution microscopy. Representativemaximum-intensity projections of merged p34 (red), F-actin (green) andGM130 (blue) are shown at low magnification. The white boxes indicatethe enlarged Golgi regions. Single focal planes of p34 and F-actin in theGolgi region are shown at high magnification. The scale bar represents10 µm. (b) HeLa cells were transfected with p34-A siRNA, Myo1b siRNAor control siRNA, immunolabelled with anti-MPR antibody and analysedfor the distribution of MPR by epifluorescence microscopy. The scalebar represents 10 µm. (c) The percentage of MPR fluorescence in theTGN region was quantified as described in the Methods (n = 3,N = 62

control-siRNA-treated cells, N = 45 p34-A-siRNA-treated cells, N = 68Myo1b-siRNA-treated cells; ∗P <0.001, ANOVA, mean±s.e.m.). (d) HeLacells were transfected with p34-A siRNA or control siRNA, and witha plasmid encoding GFP–MPR. GFP–MPR carriers were monitored at37 ◦C by time-lapse imaging using spinning-disc confocal microscopy(see Supplementary Movie S8). The first frames of representative moviesare shown at low magnification. The white boxes indicate the enlargedTGN regions. Note the tubules containing GFP–MPR (arrows). The scalebar represents 10 µm. (e) The average number of cytoplasmic carriersper frame was quantified as described in the Methods (n = 2,N = 18control-siRNA-treated cells, N = 18 p34-A-siRNA-treated cells, mean).(f) The number of tubules that formed and underwent scission perminute was quantified (n =2,N =18 control-siRNA-treated cells, N =18p34-A-siRNA-treated cells, mean).

Reduction of F-actin foci on depletion of the Arp2/3 complexphenocopies Myo1b knockdownOur data indicate that Myo1b regulates together with F-actin foci theformation of tubular-carrier precursors. According to this hypothesiswe predicted that the reduction of F-actin foci in the TGN region onArp2/3-complex knockdown should phenocopy the accumulation ofMPR in the TGN observed on Myo1b knockdown. Two p34 siRNAsdecreased the amount of the Arp2/3 complex expressed in HeLa cells asjudged byWestern blot whereasMyo1b siRNA did not affect the expres-sion level of p34. p34-A siRNA, being slightlymore efficient, was used insubsequent experiments (Supplementary Fig. S8a,b). The F-actin fociin the vicinity of the Golgi complex were no longer present in p34-A-siRNA-treated cells (Fig. 7a). Similarly toMyo1b-siRNA-treated cells,p34-A-siRNA-treated cells showed an important increase in the amountof fluorescence corresponding to the TGN pool of MPR (Fig. 7b,c).Furthermore, knockdown of the Arp2/3-complex expression reducedby 51% the number of MPR carriers and by 47% the formation of tubu-lar precursors (Fig. 7d–f and Supplementary Movie S8). We concludedthat the inhibition of actin dynamics and thereby the formation ofF-actin foci by Arp2/3-complex depletionmimics the effect of depletionofMyo1b in the biogenesis ofMPR post-Golgi carriers.

DISCUSSIONMorphogenesis of TGN transport carriers comprises the formationof tubular-carrier precursors and their extension and fission. Actindynamics and various proteins related to the actin-based system havebeen implicated in clathrin-dependent MPR and clathrin-independentp75 carrier biogenesis. However, the actin structures that participatein this function were not identified2,5,18. Here we show that absenceof F-actin foci on depletion of the Arp2/3 subunit p34 inhibitsthe formation of tubular-carrier precursors and post-Golgi carriers.Furthermore, we provide evidence that the inhibition of MPR and p75exit from the TGN onMyo1b depletion is accompanied by a decrease inF-actin foci. Together, these observations support a role of the F-actinfoci and the Arp2/3 complex in the post-Golgi traffic for the formationof tubular-carrier precursors independently of the recruitment of theclathrin coat machinery.Our results demonstrate that a pool of Myo1b located in the

TGN region controls the TGN exit of MPR and p75 but not of aGPI-anchored protein, independently of its role along the endocyticpathway, and that MPR tubular-carrier precursors, post-Golgi carriersand F-actin focus formation depend on Myo1b expression. Theseobservations together argue that Myo1b functions with F-actin foci for

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Figure 8Model for the role of Myo1b in the formation of tubular carriersat the TGN. (a) By actively tethering and orienting the polymerizingF-actin to the TGN membrane Myo1b may increase its stability and thusinduce the formation of F-actin foci. In addition, active Myo1b wouldgenerate a force to deform the TGN membrane. Myo1b is representedbefore (dashed blue line) and after (solid blue) the power stroke, duringwhich it pulls the actin filament (initial position, dashed grey line;final position, solid grey line) towards the cytosol (arrows indicatethe directions of F-actin and Myo1b movements) (1). By deformingthe membrane Myo1b would facilitate the kinesin function to extendmembrane tubular precursors along microtubules (MT, 2). Finally, thescission machinery, which includes dynamin and/or myosin 2 and

F-actin, will lead to the formation of post-Golgi tubular carriers (3).(b) The absence of Myo1b or the inhibition of its motor activity wouldnot properly orient polymerizing actin at the TGN membrane and wouldrender F-actin unstable, leading to a reduction of F-actin foci. WithoutMyo1b activity and F-actin foci, the formation of post-Golgi carrierswould be inhibited and the exit of cargo and TGN morphology wouldbe impaired. (c) The excess of Myo1b would increase the number ofsites where actin polymerization is stabilized to form more F-actinfoci. Consequently, sites where Myo1b induces membrane deformationwould increase and extra tubular precursors pulled by kinesins wouldbe detected due to the rate-limiting scission machinery. Note that thescale of the different panels of the model is not the same.

the formation of tubular-carrier precursors leading to the TGN exit oftransmembrane proteins. In contrast, membrane deformation leadingto the TGN exit of GPI-anchored proteins that distribute with lipidrafts may depend on lipid segregation28,29.How does Myo1b–F-actin promote the formation of tubular-carrier

precursors? The pulling force generated by microtubule-associatedmotors such as kinesins has been shown previously to drive theextension of tubular-carrier precursors19,30. As Myo1b–F-actin fociare required for tubular-precursor formation it is likely that theyfunction before kinesins. An attractive possibility based on Myo1bmechanochemical properties is that Myo1b–F-actin foci control TGNmembrane tension31,32 thereby promoting membrane deformation.Membrane deformation at the TGN driven by Myo1b with F-actinwould facilitate the function of a kinesin to extend tubular-carrierprecursors (Fig. 8). Indeed, it has been shown that the density ofkinesins required at the tip of a membrane tubule to extend it dependson the membrane tension33.Two other myosin 1 proteins contribute to membrane deformation.

The motor activity of Myo5p in budding yeast is required to orientpolymerizing F-actin and to invaginate the plasma membrane, leadingto endocytosis34. Myosin 1c in Xenopus oocytes controls through itsmotor domain the orientation of F-actin polymerization towards themembrane and so generates force to compress the membrane duringcompensatory endocytosis25. Our finding that Myo1b motor activityis required for the formation of F-actin foci and for the steady-stateMPR distribution indicates that, similarly to these myosins, Myo1bactively tethers and orients polymerizing F-actin to the TGNmembrane

and generates a force leading to TGN membrane deformations. Inaddition, the Myo1b motor domain may stabilize newly polymerizedF-actin, leading to the formation of F-actin foci. Similarly, myosin VIstabilizes F-actin cone structures during spermatid individualization inDrosophila and cortical F-actin inmammalian cells35–37.A growing number of myosins function at the TGN exit in

mammalian cells. Myosin 18a is required to maintain the Golgistructure38 and myosin 2 is required for scission of Rab6 tubulesemanating from the TGN (ref. 9). We confirmed that inhibition ofATPase activity of myosin 2 inhibits scission of MPR tubular-carrierprecursors, whereas our data indicate that Myo1b initiates theirformation (data not shown). Our results with these recent reportsprovide an emerging concept that different myosins are associated withthe TGN function in distinct trafficking pathways or in different stepsof the same pathway. The known structural and biochemical propertiesof these myosins strongly indicate that they function by using distinctmolecular mechanisms.Altogether, our current and previous work reveals a new function

for Myo1b, that is, to couple actin assembly to organelles and controlmembrane deformation, leading to the formation of transport carriers.Understanding the molecular mechanism by which Myo1b can achievethis function is an exciting future challenge. �

METHODSMethods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturecellbiology

Note: Supplementary Information is available on the Nature Cell Biology website

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ACKNOWLEDGEMENTSWe thank L. Salas-Cortes for designing the Myo1b siRNA, M. Prospéri for Myo1bantibodies and the Cherry–Myo1b, FlagHA–Myo1b-5MR and FlagHA–Myo1b-5ME constructs and J. Lee-Tin-Wah andP.Martin for helping set up the actin slidingassay. We thank V. Fraisier, J-B. Sibarita, F. Waharte and L. Sengmanivong for theirexpertise in microscopy and the Nikon imaging centre@Institut Curie-CNRS. Wethank E. Derivery for setting up the Myo1b purification with the FlipIn system. Wethank J. Kean for critical reading of the manuscript. This work has been supportedby the Institut Curie, the CNRS and the Agence Nationale pour la Recherche(grant ANR 09-BLAN-0027). C.G.A. has been the recipient of an EMBO long-termfellowship (ALTF 607-2006) and aMarie Curie action intra-European fellowship forcareer development (FP7-PEOPLE-2007-2-1-IEF N. 2200088).

AUTHOR CONTRIBUTIONSC.G.A. and E.C. conceived the project and wrote the manuscript; C.G.A. generatedand analysed most of the data; A.Y. carried out Myo1b and Myo1b mutantpurification as well as their characterization in vitro; D.T. and G.R. generated andanalysed the electron microscopy data; D.L. revised the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturecellbiologyReprints and permissions information is available online at http://www.nature.com/reprints

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26. Friedman, A. L., Geeves, M. A., Manstein, D. J. & Spudich, J. A. Kineticcharacterization of myosin head fragments with long-lived myosin.ATP states.Biochemistry 37, 9679–9687 (1998).

27. Shimada, T., Sasaki, N., Ohkura, R. & Sutoh, K. Alanine scanning mutagenesisof the switch I region in the ATPase site of Dictyostelium discoideum myosin II.Biochemistry 36, 14037–14043 (1997).

28. Klemm, R. W et al. Segregation of sphingolipids and sterols duringformation of secretory vesicles at the trans -Golgi network. J. Cell Biol. 185,601–612 (2009).

29. Polishchuk, R., Di Pentima, A. & Lippincott-Schwartz, J. Delivery of raft-associated,GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytoticpathway. Nat. Cell Biol. 6, 297–307 (2004).

30. Roux, A. et al. A minimal system allowing tubulation with molecular motors pullingon giant liposomes. Proc. Natl Acad. Sci. USA 99, 5394–5399 (2002).

31. Laakso, J. M., Lewis, J. H., Shuman, H. & Ostap, E. M. Myosin I can act as amolecular force sensor. Science 321, 133–136 (2008).

32. Laakso, J. M., Lewis, J. H., Shuman, H. & Ostap, E. M. Control of myosin-I forcesensing by alternative splicing. Proc. Natl Acad. Sci. USA 107, 698–702 (2010).

33. Leduc, C. et al. Cooperative extraction of membrane nanotubes by molecular motors.Proc. Natl Acad. Sci. USA 101, 17096–17101 (2004).

34. Sun, Y., Martin, A. C. & Drubin, D. G. Endocytic internalization in budding yeastrequires coordinated actin nucleation and myosin motor activity. Dev. Cell 11,33–46 (2006).

35. Naccache, S. N., Hasson, T. & Horowitz, A. Binding of internalized receptors to thePDZ domain of GIPC/synectin recruits myosin VI to endocytic vesicles. Proc. NatlAcad. Sci. USA 103, 12735–12740 (2006).

36. Noguchi, T., Lenartowska, M. & Miller, K. G. Myosin VI stabilizes an actinnetwork during Drosophila spermatid individualization. Mol. Biol. Cell 17,2559–2571 (2006).

37. Noguchi, T., Lenartowska, M., Rogat, A. D., Frank, D. J. & Miller, K. G. Propercellular reorganization during Drosophila spermatid individualization dependson actin structures composed of two domains, bundles and meshwork, thatare differentially regulated and have different functions. Mol. Biol. Cell 19,2363–2372 (2008).

38. Dippold, H. C. et al. GOLPH3 bridges phosphatidylinositol-4-phosphate andactomyosin to stretch and shape the Golgi to promote budding. Cell 139,337–351 (2009).

39. Veigel, C. et al. The motor protein myosin-I produces its working stroke in two steps.Nature 398, 530–533 (1999).

NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 789

METHODS DOI: 10.1038/ncb2262

METHODSCell culture and transfection. HeLa cells were cultured in DMEM (Invitrogen) aspreviously described40. Cells were transfected with 10–25 nM specific siRNA usingLipofectamine RNAiMax (Invitrogen) and analysed after 48 h, or with 0.5–2 µg ofcomplementary DNA using Lipofectamine 2000 (Invitrogen) and analysed after12–24 h. When indicated, cDNA was transfected after 24 h of siRNA treatmentand cells were analysed after 12–24 h. For live-cell imaging, HeLa cells were grownin glass-bottomed dishes (Fluorodish, World Precision Instruments). Cells wereimaged in DMEM without phenol red, supplemented with 10% FCS at 37 ◦C.

siRNA. Home-designed Myo1b siRNA (Myo1bH siRNA, 5′-GCTTACCTGGAAA-TCAACAAG-3′) (Sigma Proligo), On-Target Plus Myo1b-D3 siRNA (5′-CCAUAUAUGAUCCUCGAAA-3′), Myo1b-D4 siRNA (5′-GCCCGAAUCU-CGAGUGAAU-3′), p34-A siRNA (CCA UGU AUG UUG AGU CUA AUU) andp34-C siRNA (GGA CAG AGU CAC AGU AGU CUU) (Dharmacon); and anon-targeting sequence designed by Dharmacon used as control siRNA.

Plasmids. GFP–Myo1b (ref. 12); Cherry–Myo1b, prepared by subcloningGFP–Myo1b into mCherry plasmid at EcoR1; FlagHA–Myo1b, generated by sub-cloning GFP–Myo1b into FlagHA–pcDNA5 at FseI and AscI; FlagHA–Myo1b-5M,generated by site-directed mutagenesis of the plasmid encoding Flag-HA–Myo1bwith five silent mutations introduced in the siRNA target sequence (GCC TAT CTAGAG ATT AAC AAG); FlagHA–Myo1b-5ME and FlagHA–Myo1b-5MR generatedby site-directed mutagenesis of the plasmid encoding FlagHA–Myo1b-5M withthe introduction of a E490K and a N160A mutation respectively; GFP–MPR (B.Hoflack, Dresden University of Technology); chMPR (P. Lobel, University ofMedicine and Dentistry of New Jersey)41; LifeAct–Cherry (G. Montagnac, InstitutCurie)16; p16–Cherry (E. Derivery, Institut Curie); p75–GFP (G. Kreitzer, CornellUniversity)19; GFP–GPI (G. Egea, Universitat de Barcelona)21.

Antibodies. Anti-Myo1b polyclonal antibody (pAb; 1:200; ref. 12); anti-MPRmonoclonal antibody (mAb; 1:200, 2g11, Abcam); anti-chMPR-mAb (1:100; 86f7,The Developmental Studies Hybridoma Bank); anti-TGN46-pAb (1:100; Serotec);anti-GM130-mAb (1:500; BD Biosciences); anti-p34-pAb (1:200; Millipore); anti-CathepsinD-pAb (1:100; Upstate); anti-HA-mAb (1:400; 3f10; Roche); anti-tubulin-mAb (1:5,000; Sigma-Aldrich); Alexa- and horseradish peroxidase-conjugatedsecondary antibodies (1:500 Invitrogen and 1:5,000 Jackson ImmunoResearchLaboratories, respectively); anti-MPR-pAb (1:1,000; gift from B. Hoflack), anti-SNX-mAb (1:200; BD Biosciences), anti-CathepsinD-pAb (immunoprecipitate 5 µl,western blot 1:1,000; Calbiochem). Alexa-conjugated phalloidin was used to detectF-actin (1:500; Invitrogen).

Immunoblotting. Proteins separated by SDS–polyacrylamide gel electrophoresiswere transferred to nitrocellulose membranes and processed for immunoblottingusing Super Signal West Pico Chemiluminescent substrate (Pierce Biotechnology).Images of immunoblots were captured with a Fuji LAS-3000 (Fujifilm) within thelinear range and quantified by densitometry using the ‘analyse gels’ function inImageJ (http://rsb.ingo.nih.gov/ij/).

Immunofluorescence labelling. Cells were fixed with 3% paraformaldehyde42

and permeabilized with 0.3% Triton X-100 before antibody incubation using thestandard procedure. For imaging Myo1b in the perinuclear region, cells werepre-extracted with 0.3% Triton X-100 in 3% PFA for 30 s before fixation. Forimaging MPR endocytosis, cells were incubated for 5min at 37 ◦C with anti-chMPRantibodies before fixation43. For imaging p34 puncta, cells were pre-extracted with0.3% Triton X-100 in 3% PFA for 90 s before fixation. For imaging MPR, 0.3%Triton X-100 was replaced by 0.1% saponin.

Image acquisition. Epifluorescence microscopy was carried out with a LeicaDM6000B microscope equipped with a 40× NA 1.25 oil immersion objectiveand a CoolSnap HQ camera (Photometrics). Three-dimensional deconvolutionmicroscopy was carried out using a Nikon Eclipse-90i microscope equipped witha 100×NA 1.4 oil immersion objective, a piezo-electric driver mounted underneaththe objective and a CoolSnap HQ2 camera. z series of images were taken at 0.2 µmincrements. Deconvolutionwas carried out by the three-dimensional deconvolutionMetamorph module with the fast iterative constrained PSF-based algorithm44.Confocal microscopy was carried out with a Leica-TCS-SP2 equipped with a 100×NA 1.4 oil immersion objective. Spinning-disc confocal microscopy was carried outwith a Yokogawa CSU-22 spinning-disc head on a Nikon TE-2000U microscopeequipped with a 40× NA 1.3 and a 100× NA 1.4 oil immersion objective and aCoolsnap HQ2 camera, a NanoScanZ piezo focusing stage (Prior Scientific) anda motorized scanning stage (Marzhauser). These microscopes were steered withMetamorph 7.1 (Universal Imaging Corporation).

Quantitative analysis. For the quantification of MPR, chMPR, cathepsin D andLAMP1 distribution (Figs 2b, 3b,d,f, 6b and Supplementary Figs S2e and S4e)

maximum projections of z stacks were carried out if images were acquired byconfocalmicroscopy, and two regions, based on the cell boundary and on theTGN46labelling (TGN region), were outlined using the ImageJ ‘polygon selection’ tool.The fluorescence corresponding to the proteins was automatically thresholded andthe average fluorescence of each region was quantified with the ImageJ ‘Measure’function. The fluorescence in the TGN region was calculated as a percentage of thetotal cell fluorescence. For the quantification of the number of GFP–MPR carriers(Figs 4b and 7e), each carrier was identified after background correction by objectsegmentation using the wavelet-based multidimensional analysis software (MIAimaging platform; PICT-IBISA; ref. 45); the number of objects in the cytoplasm wascounted automatically using the ImageJ ‘Analyse Particles’ function.

For the quantification of the number of overlapping puncta (Fig. 5d), MPRcarrier and F-actin foci in the TGN region were identified as described above,the overlapping pixels between the two structures was calculated using the ImageJfunction ‘AND’ and the number of puncta of overlapping pixels was counted in theTGN region using ‘Analyse Particles’. The number of LifeAct–Cherry puncta in theTGN region (Fig. 5c) was counted in segmented LifeAct–Cherry movies.

For quantification of the number of F-actin foci in the Golgi region (Fig. 5f),the Golgi region was first outlined on the plane showing more GM130 staining;then the F-actin foci were thresholded and their number was determined with‘Analyse Particles’. The three-dimensional quantification of the number of p34puncta (Figs 5h and 6d) was carried out as above except that the number of objectswas counted in the three-dimensional TGN–Golgi region using the plug-in three-dimensional object counter (Fabrice Cordelières, Institute Curie). The number ofp34 puncta at the ventral plasmamembrane plane (Figs 5i and 6e) was counted with‘Analyse Particles’.

STxB uptake. Cells were incubated on ice for 30min with Alexa555-STxB or STxB(L. Johannes, Institut Curie) and chased for 1 h. After fixation and immunolabellingwith anti-GM130-mAb, STxB distribution was quantified as described for MPR.

TGN exit. Cells were grown in a two-well silicone chamber on glass-bottomeddishes. A TGN block was carried out as described previously19. TGN exit wasmonitored by time-lapse fluorescence microscopy using the spinning-disc confocalmicroscope. Aminimum of ten three-dimensional stacks (the z interval was 0.5 µm)was acquired for each condition at 10min intervals (100–200ms exposures). Toquantify protein–GFP fluorescence that remained associated with the Golgi–TGN,maximum-intensity projections were carried out using ‘stack z-project’; theTGN region at time zero was thresholded; the average fluorescence intensity ofprotein–GFP in the TGN region was measured at each time; the fluorescencemeasured at time zero was normalized to 100%.

Cathepsin D processing. Cells were pulsed for 2 h at RT using 0.1mCiml−1 [35S]methionine–cysteine (Perkin Elmer) and chased for 1, 3 and 5 h at 37 ◦C as describedpreviously46. Then, cells were lysed in 50mM Tris, pH 7.5, 150mM NaCl, 10%glycerol, 5mMEDTA, 1%TritonX-100 (vol/vol), and a protease inhibitor cocktail47

(PIC; Sigma-Aldrich). The lysates were immunoprecipitated with anti-CathepsinD antibody and analysed by SDS–polyacrylamide gel electrophoresis. Fluorographywas carried out with Typhoon 9200 PhosphorImager (Amersham Biosciences) andquantification was carried out using ImageJ.

Quantification of p75 at the cell surface. After TGN block and incubation at37 ◦C for 0, 30, 60 and 240min, cells were immunolabelled with an antibodyagainst the extracellular epitope of p75 on ice, fixed and analysed by epifluorescencemicroscopy. The integrated fluorescence intensities corresponding to p75–GFP andanti-p75 antibody were measured. p75 at the cell surface was calculated as the ratioof anti-p75/p75–GFP intensities.

β-hexosaminidase secretion assay. Cells were incubated with phenol red-freeDMEM for 8 h (ref. 48). Secreted β-hexosaminidase activity was measuredusing 5mM nitrophenyl-N -acetyl-β-D-glucosamide (Sigma-Aldrich). Release ofp-nitrophenol was measured by absorbance at 405 nm.

Electronmicroscopy. Cells were grown on coverslips, fixed with 2.5% glutaralde-hyde in 0.1 M cacodylate for 24 h and processed as described previously11. Forimmuno-electron microscopy, cells grown on coverslips were fixed in 2% PFA, in0.1M phosphate buffer at pH 7.4 and processed for ultracryotomy and immunogoldlabelling using protein A conjugated to 10 nm gold49. Sections were observed withan electronmicroscope (Philips CM120; FEI company) and digital acquisitions weremade with a numeric camera (Keen View; soft imaging system).

In vitro actin motility assay. To purify FlagHA–Myo1b, FlagHA–Myo1b-5MEor FlagHA–Myo1b-5MR: Hek293-FlipIn cells expressing FlagHA–Myo1b or thetwo mutants were lysed in buffer H (50mM Tris-HCl at pH 7.5, 150mM KCl,4mM MgCl2, 1mM EGTA, 4mM ATP, 1mM DTT, PIC) supplemented by 1%Triton X-100 according ref. 50. Lysates were incubated with anti-Flag beads

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(Sigma-Aldrich) for 2 h at 4 ◦C. Recombinant proteins were eluted with 3X FLAGPeptide (Sigma-Aldrich) and dialysed against buffer H containing 10% glycerol.

F-Actin: Rabbit skeletal muscle actin (cytoskeleton) was polymerized in buffer F(50mM Tris-HCl at pH 7.4, 50mM KCl, 1mMMgCl2, 0.2mM CaCl2 and 0.5mMDTT) for 2 h and incubated with Alexa594-phalloidin for 1 h.

Motility assay: Glass flow-cells were prepared with a nitrocellulose-coated slide.0.08 µM anti-Myo1b in 50mM imidazole-HCl at pH7.4, 25mM KCl, 4mMMgCl2,1mM EGTA, 9mgml−1 glucose, 0.4mgml−1 glucose oxidase, 0.1mgml−1 catalaseand 40mM DTT was added to the flow-cell, followed by 0.3 µM FlagHA–Myo1bor the two mutants and 0.0105 µM of F-actin filaments stabilized with Alexa594-phalloidin. Image acquisition was begun after 5min incubation with 2mM ATP at1 f/10 s.

Statistical analysis. Statistical significance was determined by Student’s t -test fortwo sets of data with Excel. For multiple comparisons we used ANOVA withSigmaPlot 11.0.

40. Loubery, S. et al. Different microtubule motors move early and late endocyticcompartments. Traffic 9, 492–509 (2008).

41. Chen, H. J., Remmler, J., Delaney, J. C., Messner, D. J. & Lobel, P. Mutationalanalysis of the cation-independent mannose 6-phosphate/insulin-like growth factorII receptor. A consensus casein kinase II site followed by 2 leucines near the carboxylterminus is important for intracellular targeting of lysosomal enzymes. J. Biol. Chem.268, 22338–22346 (1993).

42. Grosshans, B. L. et al. TEDS site phosphorylation of the yeast myosins I is requiredfor ligand-induced but not for constitutive endocytosis of the G protein-coupledreceptor Ste2p. J. Biol. Chem. 281, 11104–11114 (2006).

43. Lin, S. X., Mallet, W. G., Huang, A. Y. & Maxfield, F. R. Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recyclingcompartment en route to the trans -Golgi network and a subpopulation of lateendosomes. Mol. Biol. Cell 15, 721–733 (2004).

44. Sibarita, J. B. Deconvolution microscopy. Adv. Biochem. Eng. Biotechnol. 95,201–243 (2005).

45. Racine, V. et al. Visualization and quantification of vesicle trafficking on athree-dimensional cytoskeleton network in living cells. J. Microsc. 225,214–228 (2007).

46. Mardones, G. A. et al. The trans -Golgi network accessory protein p56 promoteslong-range movement of GGA/clathrin-containing transport carriers and lysosomalenzyme sorting. Mol. Biol. Cell 18, 3486–3501 (2007).

47. Piccirillo, R. et al. An unconventional dileucine-based motif and a novel cytosolicmotif are required for the lysosomal and melanosomal targeting of OA1. J. Cell Sci.119, 2003–2014 (2006).

48. Riederer, M. A., Soldati, T., Shapiro, A. D., Lin, J. & Pfeffer, S. R. Lysosomebiogenesis requires Rab9 function and receptor recycling from endosomes to thetrans -Golgi network. J. Cell Biol. 125, 573–582 (1994).

49. Mallard, F. et al. Direct pathway from early/recycling endosomes to the Golgiapparatus revealed through the study of shiga toxin B-fragment transport. J. CellBiol. 143, 973–990 (1998).

50. Derivery, E., Lombard, B., Loew, D. & Gautreau, A. The wave complex is intrinsicallyinactive. Cell Motil. Cytoskeleton. 66, 777–790 (2009).

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Figure S1 F-actin foci colocalize with the Arp2/3 complex in the perinuclear region. The distribution of the Arp2/3 complex in HeLa cells detected by immunofluorescence with anti-p34 antibody and the distribution of F-actin detected by fluorescent phalloidin were analysed by 3D deconvolution microscopy. A representative maximum intensity

projection of merged p34 (green) and F-actin (red) distribution at low magnification and single focal medial plane of F-actin and p34 merged and alone at high magnification are shown. The white box indicates the enlarged perinuclear region. Scale bar represents 10 µm.

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Figure S2 MPR distribution upon Myo1b KD or over-expression. (a) HeLa cells were transfected with siMyo1bH, siMyo1bD3 and siMyo1bD4, or siControl, and the expression of Myo1b was analysed by immunofluorescence or (b) by Western blotting with anti-Myo1b antibody (Scale bar represents 10 µm). Tubulin was used as loading control. (b) The doublet detected with anti-Myo1b corresponds to the two Myo1b alternative spliced isoforms detected in HeLa cells. The uncropped Western blot is shown in Fig. S9. (c) The amount of Myo1b detected by Western blot was quantified as described in the material and methods (n=3, *p< 0.001, Anova, mean±SEM). (d) HeLa cells were transfected with siMyo1bH, siMyo1b D3, siMyo1b D4 or siControl, immunolabelled with anti-MPR and anti-TGN46 antibodies, and analysed by epifluorescence microscopy. Scale bar represents 10 µm. (e) The percentage

of fluorescence corresponding to MPR in the perinuclear region was quantified as described in the material and methods (n=2, N=22 siControl-treated cells, N=27 siMyo1b-treated cells, N=30 siMyo1bD3-treated cells, N=30 siMyo1bD4-treated cells, mean). (f, g) HeLa cells were transfected with FlagHA-Myo1b or without (Mock), immunolabelled with anti-MPR (green), anti-HA (inset), and anti-SNX1 (red) or (g) anti-TGN46 (red) antibodies, and analysed by 3D microscopy. Representative focal planes are shown. White boxes indicate the enlarged region that is shown after deconvolution. Note the similar overlap between MPR and SNX1 in Mock and FlagHA-Myo1b-expressing cells. Although the TGN is more scattered when Myo1b is overexpressed an overlap of TGN46 with MPR signal is still observed. Scale bars represent 10 µm.

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Figure S3 Retrograde transport of STxB in cells transfected with control or Myo1b siRNA. (a) HeLa cells transfected with siControl or siMyo1b were incubated with Alexa555-STxB (colour coded green) for 30 min on ice, washed and chased during the indicated time at 37°C before fixation. Then cells were immunolabelled with anti-GM130 antibody (red) and analysed by epifluorescence microscopy. Scale bar represents 10 µm. (b) The percentage

of fluorescence corresponding to STxB in the perinuclear region was quantified as a function of time and as described in material and methods (n=2, N=20-60 cells siControl-treated cells, N=18-56 siMyo1b-treated cells mean±SEM. (c) HeLa cells were transfected with siControl or siMyo1b, incubated with STxB (60 min), analysed by immuno-EM. Note that STxB localizes to the TGN both in siControl and siMyo1b-treated cells. Scale bars represent 250 nm.

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Figure S4 Processing and distribution of cathepsin D, distribution of LAMP1 and p75-GFP delivery to the plasma membrane in cells transfected with control or Myo1b siRNA. (a) HeLa cells transfected with siControl or siMyo1b were metabolically labelled for 2 h at 20°C with [35S] methionine and chased for the indicated times at 37°C. Immunoprecipitation of cathepsin D was analysed by SDS-PAGE and fluorography. P: precursor, I: intermediate form and M: mature form. The uncropped gel is shown in Fig.S9 (b) The level of the intermediate form was quantified relative to the total cathepsin D levels as described in material and methods and represented as a function of time (n=2, mean). (c) HeLa cells were transfected with siControl or siMyo1b, incubated overnight with rhodamine-dextan (red) at 37°C before fixation immunolabelled with anti-cathepsin D antibodies (green) and

analysed by 3D deconvolution microscopy. The representative focal planes of the TGN region are shown. Scale bar represents 10 µm. The percentage of colocalisation indicated in the merged image (yellow) was quantified as described in the material and methods. (d) HeLa cells were transfected with siControl or siMyo1b and immunolabelled with anti-LAMP1 antibodies. Scale bar represents 10µm. (e) The percentage of fluorescence corresponding to LAMP1 in the perinuclear region was quantified as described in material and methods (n=2, N=14 siControl-treated cells, N=20 siMyo1b-treated cells, mean). (f) HeLa cells transfected with siControl or siMyo1b and plasmids encoding p75-GFP were immunolabelled with anti-p75 antibody at 0 and 180 min of chase, fixed and analysed by 3D microscopy. Representative focal planes are shown. Scale bar represents 10 µm.

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Figure S5 MPR tubules induced by Myo1b over-expression contain TGN46. HeLa cells co-transfected with plasmids encoding GFP-MPR (green) and FlagHA-Myo1b or with GFP-MPR alone were quickly fixed (2 min), immunolabelled with anti-HA and anti-TGN46 antibodies (red)

and analysed by 3D microscopy. Representative focal planes at low and high magnifications are shown. Note that in FlagHA-Myo1b-expressing cell the tubules contain TGN46. Scale bar represents 10 µm.

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Figure S6 Myo1b over-expression alters F-actin organization. (a) HeLa cells transfected with siControl or siMyo1b were labelled with fluorescent phalloidin and analysed by epifluorescence microscopy. Scale bar represents 10 µm. (b) The number of cells with many (as shown for siControl and siMyo1b-treated cells) or few actin stress fibres (not shown) was counted and normalized to the total cells analysed in 6 independent experiments (n=6, N=265 siControl-treated cells, N=264 siMyo1b-treated cells, % of cells).

(c) HeLa cells were transfected with plasmids encoding Cherry or Cherry-Myo1b, labelled with fluorescent phalloidin and analysed by epifluorescence microscopy. Scale bars represent 10 µm. (d) The number of cells with many (as shown for Cherry-expressing cells) or with few (as shown for Cherry-Myo1b-expressing cells) actin stress fibres was counted and normalized to the total cells analysed in 2 independent experiments (n=2, N=103 Cherry-expressing cells, N=95 Cherry-Myo1b-expressing cells; % of cells).

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Figure S7 Myo1b mutant activity and their cellular distribution. (a) Purified FlagHA-Myo1b, FlagHA-Myo1b-5MR or FlagHA-Myo1b-5ME recombinant proteins were incubated with F-actin stabilized by Alexa594-phalloidin and monitored by time-lapse imaging upon ATP addition. Merged images of actin filaments at time 0 (green) and time-projections of 25 sec (red), indicating the trajectory of the filaments. The average velocity of actin filaments is indicated in each panel. Scale bar represents 10 µm. Note that red F-actin tracks are

observed only in presence of FlagHA-Myo1b. (b) HeLa cells were transfected with plasmids encoding FlagHA-Myo1b-5M, FlagHA-Myo1b-5MR or FlagHA-Myo1b-5ME, immunolabelled with anti-HA antibody and analysed by 3D deconvolution microscopy. Representative maximum intensity projections at low magnification and single focal planes at the Golgi region at high magnification of HA are shown. Note that FlagHA-Myo1b-5M and FlagHA-Myo1b-5MR but not FlagHA-Myo1b-5E localize to the Golgi region. Scale bar represents 10 µm.

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MFl

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A-M

yo1b

-5M

RFl

agH

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yo1b

-5M

E

HA

z-proj Golgi region

b

Almeida Fig. S7

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Figure S8 Arp2/3 levels are efficiently depleted by siRNA. (a) HeLa cells were transfected with sip34 A and C, siMyo1b and siControl. The level of expression of p34 and Myo1b was monitored by Western blot. The uncropped

blot is shown in Fig.S9. Tubulin was used as loading control. (b) The amount of p34 detected by Western blot was quantified as described in material and methods (n=3, *p<0.05, Anova)

0

20

40

60

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120

p34

lelv

els

(%)

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Almeida Fig. S8

sip34

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siCon

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trol

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Myo1b

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Figure S9 Full scan of gel/blots that have been cropped in supplementary figures. Black squares indicated the cropped images.

83 kD

175 kD

47.5 kD

62 kD

Fig. S2b

83 kD

175 kD

47.5 kD

62 kD

32.5 kD

25 kD

32.5 kD

Fig. S8a

150 kD

250 kD

50 kD

100 kD

75 kD

37 kD

25 kD

20 kD

15 kD

10 kD

Fig. S4a

83 kD

175 kD

47.5 kD

62 kD

32.5 kD

83 kD

175 kD

47.5 kD

62 kD

32.5 kD

25 kD

WBPonceau S

WBPonceau S

Myo1b

Myo1b

Tubulin

Tubulin

p34

Ameida Fig.S9

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Supplementary Movie Legends

Movie 1 Representative movies of GFP-MPR exit from TGN in siControl- and siMyo1b-treated cells. The kinetics of exit of GFP-MPR from TGN was monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy in siControl- and siMyo1b-treated HeLa cells after its accumulation in the Golgi complex upon incubation at 20°C for 2 h. Simultaneous multi-position 3D images stacks were acquired at 1 stack/10 min and representative maximum intensity projections are shown at 4 f/sec. Scale bar represents 10 µm. Fig. 3B is extracted from these movies.

Movie 2 Representative movies of p75-GFP exit from TGN in siControl- and siMyo1b-treated cells. The kinetics of exit of p75-GFP from TGN was monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy in siControl- and siMyo1b-treated HeLa cells after its accumulation in the Golgi complex upon incubation at 20°C for 2 h. Simultaneous multi-position 3D images stacks were acquired at 1 stack/10 min and representative maximum intensity projections are shown at 4 f/sec. Scale bar represents 10 µm.

Movie 3 Representative movies of GFP-GPI exit from TGN in siControl- and siMyo1b-treated cells. The kinetics of exit of GFP-GPI from TGN was monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy in siControl- and siMyo1b-treated HeLa cells after its accumulation in the Golgi complex upon incubation at 20°C for 2 h. Simultaneous multi-position 3D images stacks were acquired at 1 stack/5 min and representative maximum intensity projections are shown at 4 f/sec. Scale bar represents 10 µm.

Movie 4 Representative movies of GFP-MPR dynamics in siControl- and siMyo1b-treated cells. HeLa cells were transfected with siMyo1b or siControl, and with a plasmid encoding GFP-MPR. GFP-MPR carriers were monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy. A confocal section throughout the nucleus was acquired at 1 f/sec during 1 min are shown at 15 f/sec. Scale bar represents 10 μm. Fig. 4A is extracted from these movies.

Movie 5 Representative movie of Cherry-Myo1b and GFP-MPR dynamics. The dynamics of Cherry-Myo1b and GFP-MPR was monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy in HeLa cells. A confocal section throughout the nucleus was acquired at 1 f/sec during 16 sec and is shown at 15 f/sec. Scale bar represents 10 µm. Fig. 4B is extracted from this movie.

Movie 6 Representative movies of GFP-MPR dynamics in Mock-cells and Cherry-Myo1b-expressing cells. HeLa cells were co-transfected with plasmids encoding GFP-MPR and Cherry-Myo1b. GFP-MPR carriers were monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy. A confocal section throughout the nucleus was acquired at 1 f/sec during 1 min are shown at 15 f/sec. Scale bar represents 10 μm Fig. 4C is extracted from these movies.

Movie 7 Representative movies of GFP-MPR and LifeAct-Cerry in siControl- and siMyo1b-treated cells. HeLa cells were transfected with siMyo1b or siControl, and with plasmids encoding GFP-MPR and LifeAct-Cherry. GFP-MPR and LifeAct-Cherry were monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy. A confocal section throughout the nucleus was acquired at 1 f/sec during 1 min are shown at 15 f/sec. Scale bar represents 10 μm. Fig. 5A is extracted from these movies.

Movie 8 Representative movies of GFP-MPR dynamics in siControl- and sip34 A-treated cells. HeLa cells were transiently transfected with sip34 A or siControl, and with a plasmid encoding GFP-MPR. GFP-MPR carriers were monitored at 37°C by time-lapse imaging using spinning-disk confocal microscopy. A confocal section throughout the nucleus was acquired at 1 f/sec during 1 min are shown at 15 f/sec. Scale bar represents 10 μm. Fig. 7D is extracted from this movie.

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