Ato o orsodne([email protected]) correspondence for *Author 108, Pfotenhauerstrasse Germany. Genetics, Dresden, and 01307 Biology Cell Molecular of Institute Planck aay nvriy aeie3 uligA,650Bn,CehRepublic. Czech Brno, 62500 A1, Building 3, Kamenice University, Masaryk rse,Germany. Dresden, ial Anitei Mihaela regulate trafficking that receptor genes 6-phosphate identifies mannose screen siRNA high-throughput A ARTICLE RESEARCH ß eevd1 uy21;Acpe 3Spebr2014 September 23 Accepted 2014; July 11 Received 2003; al., et (Ghosh 1 II factor growth ligands, other insulin-like internalizes MPRs two including plasma the the also of one and MPRs where system membrane, ligands. endosomal the these between unload recycling undergo they the the where and the hydrolases, system to lysosomal of to endosomal bind trans-Golgi recycling they pathway the where the (TGN), between network on (MPRs) proper secretory receptors relies 6-phosphate the process the mannose 6-phosphate-tagged This and requires from system. mannose endosomal components autophagy synthesized, hydrolases cytosolic newly by lysosomal of of over delivery or turned signaling pathogens organelles nutrients, and endocytosed of molecules degradation lysosomal The INTRODUCTION Lysosome Trafficking, receptor, transport, 6-phosphate Post-Golgi Mannose screen, and siRNA WORDS: signaling KEY RAC1 and SRC- activity. a ARF1 II tubular through myosin regulating regulating post-Golgi system endosomal pathway of the enzymes biogenesis dependent enter the to destined on controls carriers signaling based cell reveals subcellular how it Furthermore, pathways the metabolism. the phosphate maps of phosphatidylinositol trafficking analysis states Our MPRs. dynamic MPR the by the encountered and/or compartments trafficking and RNA regulate MPR that kinases networks 127 signaling high-throughput different identified to belonging we phosphatases phosphatome. analysis, used and image kinome high-content human we Using the target to trafficking, signaling (RNAi) interference cell MPR how multicellular explore of To development pathways. modulates these the use to also processes Many production organisms biological membrane. virus from diverse plasma ranging regulating the proteins and the transmembrane endosomes trans-Golgi of the (TGN), trafficking between proper network (MPRs) the cell receptors on 6-phosphate and function relies mannose lysosome process for This essential homeostasis. to is hydrolases system lysosomal endosomal soluble the synthesized newly of delivery The ABSTRACT ei Meyenhofer Felix itcnlg etr ehiceUniversita Technische Center, Biotechnology 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,57–02doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 eateto itlg n mrooy aut fMedicine, of Faculty Embryology, and Histology of Department 1 auChenna Ramu , 3 acBickle Marc , ¨ rse,Ttbr 75,01307 47-51, Tatzberg Dresden, t 1 3 onlaCzupalla Cornelia , aioZerial Marino , 3 n enr Hoflack Bernard and 3 Max 1 ia Esner Milan , lhie’ ies Aah ta. 09 ahw tal., et 2008), Mathews al., neuronal 2009; et al., with et (Borck associated (Adachi these retardation disease been regulating mental the Alzheimer’s proteins [e.g. have to hydrolases in pathologies pathways synthesized Mutations newly transport and system. of disorders delivery endosomal of importance correct storage physiological of the the development illustrate Lysosomal the pathogenesis and bacterial organisms. fate and (Port cell Wntless regulate multicellular signaling and which 2011) or al., 2010), 1996) et other Basler, al., (Benhra of et Notch number (Alconada like a receptors proteins with viral routes e.g. trafficking 1996b). proteins, their al., share et MPRs Munier-Lehmann The 1996a; al., et Munier-Lehmann opeesv iwo o inln oeue oto MPR control a tissue obtain molecules to signaling or order how in trafficking. genome of development human kinases view the the comprehensive during of silenced phosphatases systematically the signaling mechanisms have and we the cell Here, in about homeostasis. homeostasis to organelle known and is response trafficking protein less coordinate Much that the 2007). actin compartments and target 2010), with (Pfeffer, fusion al., and their movement et sustaining for components (Hirokawa core membrane motors microtubules and molecular and F-actin 2010), bridging along into al., (McMahon et molecules (Anitei receptors membranes dynamics 2011), shaping transmembrane They while Boucrot, segregate described. during carriers been that have transport function coats system endosomal cell include their the to neuronal showing transport thereby proper 2012), a al., development. for et (Bucci Charcot-Marie- importance in disease seen as Tooth functions, neuro-muscular and 2002)] xeietlrpiae)treigec fte70human 710 three the gene, of per each siRNAs targeting three replicates) small (siRNAs; experimental with RNAs transfected were in interfering screen, cells endosomal primary to GFP–MPR-expressing a read-out in The which a trafficking peripheral 1A–C). as GFP–MPR in (Fig. used changes was TGN evaluate GFP–MPR more perinuclear (also of the distribution TGN46 in steady-state the with TGOLN2) as colocalized known and also the GFP–MPR in (ciMPR) compartments. TGN MPR cation-independent perinuclear endogenous colocalized the highly green GFP–MPR with 2003). al., the receptor et 6-phosphate (Waguri cytoplasmic mannose comprising (GFP–MPR) cation-independent and the transmembrane protein of the domain to chimeric fused protein a cells fluorescent HeLa used expressing we transport, stably MPR controlling genes identify To of trafficking regulators MPR new identifies screening siRNA-based RESULTS aymcieisfntoigi noyoi n biosynthetic and endocytosis in functioning machineries Many 2 aaChrist Sara , 1, * 3 tfiLenhard Steffi , 1 esi Korn Kerstin , 5079 3 ,

Journal of Cell Science EERHARTICLE RESEARCH 5080 ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal odtos H itiuin frobust of Distributions respective (H) the conditions. in co-labeled the markers represent organelle EEA1 and LAMP1 GM130, el bu) aaeesP oP8are P48 to P1 Parameters (blue). untreated cells and screen (red) secondary nocodazole-treated the (gray), in tested for siRNAs measured all objects) P43 GFP–MPR and of size) (number object (GFP–MPR P18 for 10 (si bars: membrane plasma the at (si tubules vra ewe F–P n GM130). (50% and P48 GFP–MPR and between nucleus) overlap the and GFP– objects between MPR (distance P24 objects), GFP– of MPR (number P43 P18 size), for object experiment (GFP–MPR entire the in measured and 003 002, targeting 001, 004) siRNAs panel, siRNA lower panel, 001; (upper siRNAs for experiments S2. Robust Fig. (G) material supplementary in described upesrkns ;si 2; kinase suppressor (si fragmentation periphery (si the in GFP–MPR included increased cells RNAi-treated and control MOCK- in observed TfR. phenotypes or GFP–MPR LAMP1 (E) GM130, antibodies AP-2, with AP-1, co-labeled against were cells screen, which secondary in the in re-tested were Selected genes phosphatases. and targeting kinases siRNAs human with incubated GFP– were expressing MPR stably cells RNAi-based HeLa the screens. of Scheme (D) condition). ofiin R)creain sn h Volocity (median the software using correlation, (Rr) correlation coefficient Pearson the calculating analyzed by was Colocalization (A) fluorescence microscopy. by analyzed anti- and (C) or TGN46 anti-ciMPR (B) with co-labeled were 1. Fig. CHEK2 AC OKtetdGPMRcells GFP–MPR MOCK-treated (A–C) m hcpitkns ) TGN 2), kinase checkpoint , .()Robust (F) m. NTRK3 Z soe optdi replicate in computed -scores INPP5J LATS2 6 n nrae GFP–MPR increased and ) s.d.; P1 P2 TfR, AP-2, AP-1, . INPP5J ag tumor large , n Z . soe calculated -scores 0clsper cells 20 INPP5J ,GFP–MPR ), .Scale ). Z -scores

Journal of Cell Science 1) asda cuuaino F–P ttecl surface cell the at GFP–MPR of accumulation an caused whose S1D), genes In of 11 that S3B). of to subgroup similar Table a knockdown, detected material screen (supplementary visual the analysis addition, as visual an confirmed by with were S3A) indicated hits Table (and material analysis supplementary image in slightly for asterisk found set threshold genes the Eight under S3A). 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(P24, (P47, nucleus markers parameters the organelle GFP–MPR to with colocalization distance measuring and P45), P25) (P43, parameters number of P19), Pearson (P18, group size a S1E,F). Fig. validated material si (supplementary measured S2B,D) repeats Fig. values experimental different parameter for the the in material between shown coefficients supplementary correlation si is 1E,G; (Fig. J), profiles polyphosphate-5-phosphatase organelle parameter MOCK- robust respective control calculating of to the by normalized cells and were treated fluorescent values nuclei the S2A) Parameter GFP–MPR, markers. of Fig. i.e. characterization material from resulting objects, comprehensive software supplementary parameters a in 48 MotionTracking coats). of provided (described set the clathrin analysis The endocytic 2010). this using a al., of et analyzed (AP-1, the component (Collinet at were 1 (a sorting MPR AP-2 Images in protein involved and coats adaptor TGN) clathrin the compartments), endosomal of late component of lysosomal associated marker a lysosomal or (as the LAMP1 (EEA1), matrix shuttles glycoprotein 1 membrane endosomes), antigen which cis-Golgi recycling endosome and (TfR, early co- early the the receptor membrane, by plasma against transferrin the S2). analyzed between the antibodies GM130, Table were with each protein material integrity cells targeting and supplementary labeling repeats) morphology 1D; experimental (Fig. Organelle six gene gene, selected (four per siRNAs stealth siRNAs with transfected were cells MPR-expressing the slightly selected to analysis. found we further distance total, hits for In their genes visually. The examined 246 and Methods). were parameters threshold objects and the number under GFP–MPR Materials the measure (see of i.e. to nucleus size distribution, the GFP–MPR analyzed and in were changes reflecting Images S1A,B,D). iha|median a with |median hs 4 ee eeraaye nascnaysre.GFP– screen. secondary a in reanalyzed were genes 246 These ee eeslce fa es w tat iNstreigthe targeting siRNAs stealth two least at if selected were Genes Robust_Z-score ewe . n .,ad1 te genes other 14 and 1.8, and 1.4 between | Robust_Z-score Robust_Z-score CLTC CLTC ewe .4 n 1.4 and 1.244 between | ]adpamclgclagents pharmacological and )] Z | . splmnaymtra Fig. material (supplementary soe.Tereproducibility The -scores. .,4 ee iha with genes 43 1.8, CLTC INPP5J n nocodazole and Z (inositol -scores ritniy(upeetr aeilFg 2;TbeS3A). Table S2A; Fig. material nucleus (supplementary the intensity to distance or number, size, object measuring parameters asdtedseso fteTRpstv perinuclear genes 40 of TfR-positive subgroup Golgi a the Golgi Interestingly, of the integrity endosomes. GM130-rich the recycling maintain the and they of that of suggesting fragmentation 2D), (Fig. dispersion the 5) and compartment subunit the regulatory DLG3 caused 3-kinase, FGFR1, (phosphoinositide of P101-PI3K) ( 3 Depletion homolog large positioning. [disks they endosomes that organelle indicating LAMP1-positive S3A), modulate Table and/or material supplementary EEA1- 2F; affected (Fig. of genes of distribution or of subgroup A degradation endosomal the markers. and/or key endosomal expression of respective These the the trafficking or S3A). the components, Table regulate endosomes lysosomal material thus 2A] supplementary Fig. might 3); genes 2D,E; Fig. si kinase 3; [e.g. kinase [e.g. (Fig. kinase EEA1-positive LAMP1-positive protein activated and/or TfR-positive, 2A] of Fig. the intensity affecting 4) si GFP– (cluster and/or of without size or the 3) size increased no (cluster 4 with in and objects, 3 resulted MPR S3B). clusters in Table 22.8% the genes material of remaining 13.9% endosomes, Depletion (supplementary of the phenotype peripheral dispersion visual of of in apparent a depletion GFP–MPR depletion the of induced whereas amount clusters compartment, the these increased GFP–MPR in 63.29% genes of perinuclear depletion the the that confirmed of analysis Visual 2A]. Fig. si and 1) receptor tat iNssoe rbs Z-score| two |robust least organelle at a if For showed significant S3B). siRNAs as considered stealth Table were material changes markers, supplementary 1E; (Fig. hm( n )caatrzdb hne nojc ubrand/or number object of in two changes by distribution, characterized GFP–MPR 2) and affecting (1 genes them of our clusters GFP–MPR four with illustrating agreement 2003). al., studies good et (Waguri in microscopy distribution was electron analysis previous in state Thus, parametrical endosomes. steady late at our were LAMP1-positive present indicating and were coefficient, overlap early LAMP1, GFP–MPR EEA1-positive or of Rr of amounts EEA1 small low and that degrees Golgi GFP–MPR distinct a between low but correlation detected but adjacent Relatively Pearson and to overlap localization GFP–MPR the subcompartments. high their 2B). by (Fig. with a overlap consistent shown 50% showed as the GM130 and TfR, organelle well Rr colocalized and different coefficient GFP–MPR cells, AP-1 the control and with In 2A). GFP–MPR affected (Fig. genes of markers selected the co-distribution how analyze the to us allowed data These analysis Phenotypic oprmn TN ra nraei h muto GFP–MPR GFP–MPR-rich of si amount perinuclear [e.g. the endosomes in the peripheral increase in an of or might (TGN) fragmentation GM130) compartment some (increased and in the 2 GFP–MPR and between and reflect number, overlap 1 and/or reduced clusters nucleus a The cases the in S2C). to genes distance Fig. the GFP–MPR material for (supplementary observed clusters respectively correlation changes in (modified 2), distribution and and Pearson number 1 object good clusters and in 3). 4), a (modified and (Fig. intensity 3 showed and size parameters object intensity between the correlation in between changes and/or by calculated 4) and coefficients (3 size two other the object and nucleus, the to distance PPP2R3A irrhclgn n aaee lseigaayi identified analysis clustering parameter and gene Hierarchical ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal poenpopaae2 euaoysbntB subunit regulatory 2, phosphatase (protein CSNK2B Drosophila csi iae2 eaplppie in polypeptide) beta 2, kinase (casein FGFR1 ]adPKR as nw as known (also PIK3R5 and )] fbols rwhfactor growth (fibroblast § MAP3K3 o n fthe of any for 2 (mitogen- 5081 0 a );

Journal of Cell Science EERHARTICLE RESEARCH 5082 ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal itnet ulu)o al rlt endosomes. late or early of nucleus) to distance (increased dispersion or clustering) to increased distance nucleus, (reduced clustering late (F) or (blue); (yellow) endosomes early of enlargement (E) endosomes (orange); TfR–positive of enlargement cis-Golgi or fragmentation (blue) (red), compartment TfR the fragmentation of (D) and only GFP–MPR (D–F) (C) or affected GFP–MPR (median depletions basis whose plate Genes per (C–F) a on MOCK- cells in treated calculated co- were the markers and organelle GFP–MPR labeling between 50% (P48) the overlap and object Rr) coefficient correlation (Pearson 10 bar: Scale S3). Table material (supplementary compartment 2-positive eue P1itniy n si and intensity, AP-1 reduced LAMP1-positive si and (E). EEA1- respectively the objects, of intensity and size h opooyo h f oprmn (D). compartment TfR si the of morphology the si and GM130- compartments, and Golgi GFP–MPR positive si the (red). of AP-2 dispersion or caused AP-1 GM130, TfR, against LAMP1, antibodies EEA1, with incubated were cells HeLa secondary GFP–MPR-expressing the siRNA-treated in screen. treatment siRNA upon distribution kinases identified phosphatases. of and analysis Phenotypic 2. Fig. PPP2R3A n si and MAP3K3 A xmlso organelle of Examples (A) m .()Tecolocalization The (B) m. EPHB2 asda nraei the in increase an caused ERBB2 EHrcpo B2) receptor (EPH CSNK2B fetdteAP- the affected 6 FGFR1 s.d.). affected

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Journal of Cell Science niheti h AK oa dein n signaling, revealed Wnt 2 adhesion, and focal 1 clusters MAPK, in the genes in the enrichment of analysis Pathway GFP–MPR- the analysis AP-1 Network of GFP– and/or homeostasis compartment. regulate the AP-1-positive or GFP–MPR specifically and/or positive they trafficking affected that MPR suggesting exclusively distribution, 2C) (Fig. ARTICLE RESEARCH 5084 q-val (FDR) rate discovery q-value false The FDR 2005). a al., indicate et Subramanian (…) 2003; Dots al., et (B) (Mootha set. (GSEA) each Analysis Enrichment for Set calculated Gene the using transport. calculated GFP–MPR was control (C) pathways Signaling 4. Fig. the and secretory the of compartments the different the of along components trafficking (PIP MPR phosphate regulating Phosphatidylinositol in phosphatases and kinases enriched Lipid 4B), was (Fig. 4C). (Fig. pathway pathway 4 of cytoskeleton metabolism components phosphatidylinositol (GPCR) cluster in actin receptor enriched the whereas was G-protein-coupled 3 of Cluster the regulation 4A). (Fig. and pathways junction adherens oiiefrGPMRadEA G,adteGli() cl as 10 object bars: GFP–MPR Scale (F) (H). microscopy. DMS Golgi control confocal the and by p38-inhibitor-treated and analyzed in (G), then measured EEA1 and values and the (red) between GFP–MPR GM130 ratios for (H) The positive or ImageJ. ( 10 using (red) with shown analyzed EEA1 or were are (G) DMSO a.u.) cells with against units, incubated (arbitrary antibodies were size with GFP–MPR and co-labeled expressing number h, Cells transport. 1 GFP–MPR for modulates SB203580 signaling p38 (F–H) screen. siRNA-based n 5 experiments, 4 . 0clsprcniin median condition, per cells 50 ytei scompartmentalized is synthesis ) AC aha niheto h ee rue ncutr n A,cutr3()adcutr4 cluster and (B) 3 cluster (A), 2 and 1 clusters in grouped genes the of enrichment Pathway (A–C) , 10 2 12 6 D ii-oiyn nye n E opnnso AKptwy dniidi the in identified pathways MAPK of components (E) and enzymes Lipid-modifying (D) . .. * s.d.; m M. P 5 .09 n-a ANOVA, one-way 0.0019, -hsht P-P neryedsms[h ls III class [the 4)], (PI-4,5P endosomes subunit 4,5-bisphosphate regulatory early phosphatidylinositol (phosphoinositide-3-kinase, on PIK3R4 of (PI-3P) phosphatidylinositol synthesis [PI4K2B beta)], 3-phosphate TGN 2 the type the catalyzing 4-kinase Table on (phosphatidylinositol kinases (PI-4P) material 4-phosphate included and supplementary phosphatidylinositol kinases These 4D; five (Fig. identified S3A,B). screen traffic phosphatases docking Our membrane 2008). as two regulating al., used components et are (Vicinanza various PIPs by specific platforms where pathways, endocytic niaigaprubto tteTN elto fthe of Depletion TGN. the compartment, at GFP–MPR a perturbation (phosphoinositide-3- perinuclear in resulted a the PIK3AP1 PI4K2B indicating of of and Depletion 1)]. fragmentation protein PIK3R5 adaptor and kinase beta), subunit 1)] catalytic 3-kinase, (phosphatidylinositol-4,5-biphosphate PIK3CB (PI-3,4,5P 5-kinase-like phosphatidylinositol-3,4,5-trisphosphate (phosphatidylinositol-4-phosphate ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal a 5 .1.Arw niaeprpea endosomes peripheral indicate Arrows 0.01). 2 m [PIP5KL1 ) 3 3 inhibitor p38 M casI [class ) O-treated ewas ue

Journal of Cell Science h iaePKC ro I3P asdteacmlto of accumulation the caused PIK3AP1 of or PIK3CB and kinase early of the INPP5J, from 5-phosphatase the transport of Knockdown MPR endosomes. recycling controls MTMR4 thus colocalized S3A,B), that Fig. suggesting it material (supplementary where MPR structures, endogenous TfR-positive with GFP– perinuclear TGN. endosomes on peripheral the EEA1-positive of and on fragmentation detectable some was caused MTMR4 also 2010) al., et (Naughtin 4) protein related (myotubularin MTMR4 3-phosphatase ARTICLE RESEARCH hwdta R yoiepopoyainwsrdcdin reduced was phosphorylation activation. tyrosine SRC proteins SRC affect that phosphotyrosine-containing we could showed kDa)], of receptors 70/80 TGFBR2 II these Immunoprecipitation receptor and whether beta 3) asked factor, type growth receptor, [NTRK3 receptors (transforming kinase, two GFP–MPR of tyrosine of depleted cells (neurotrophic number in (increased detected was phenotype tubules) same perinuclear both the the Accordingly, S3D,E). Because on Fig. 2010). material detectable (supplementary al., compartment were MPR-positive carrier et GFP–PAK3 (Anitei MPR and might TGN GFP–SRC during that the dynamics 2006) at GFP–MPR formation actin al., of et RAC1-dependent number (Baust control components are the GIT2 coat and in PAK3 by clathrin/AP-1 S3B). increase detected Table material phenotype an (supplementary unique tubules i.e. a depletion caused analysis, Interestingly, PAK3 5A). visual or (Fig. GIT2 2005) SRC, al., of et (Cdc42/Rac)-activated the (Zhang protein 3] and [p21 kinase 2) PAK3 ArfGAP kinase interacting (G- p21-activated GIT2 kinase (GAP) receptor molecules, protein protein-coupled regulatory GTPase-activating actin ARF1 downstream the including to major linked a hub was SRC kinase) signaling that tyrosine showed genes non-receptor STRING these proto-oncogene, of 4A). (SRC 2011) (Fig. al., in pathway et enrichment (Szklarczyk regulatory significant analysis actin a showed the also of 2 components and GFP–MPR 1 dynamics clusters actin-myosin in signaling and Genes tubules and GFP–MPR p38 of Biogenesis homeostasis that TGN. the endosome to suggests endosomes from recycling early This h 1 Golgi 4H). normal for modulates (Fig. a that exhibited SB203580 but objects 4F,G), morphology SB203580. GFP–MPR with (Fig. peripheral EEA1 affected inhibitor treated of with overlapped number signaling p38 cells higher signaling a the p38 control, exhibited SAPK/JNK used that to and we confirm Compared as transport, p38 To well as GFP–MPR the 4E). 2012), (Fig. dual- al., in et cascades the (Chia participating included integrity Golgi kinases These 2), control phosphatase 4A). to specificity (Fig. shown (dual DUSP2 nucleus the phosphatase and/or the specificity number of object to phosphatases GFP–MPR distance the genes and increased the among depletion kinases enriched whose particularly identified pathway, MAPK also classical screen Our pathways signaling MAPK TGN. the from trafficking MPR in metabolism lipid cone-shaped importance theof the indicating in S3C), GFP–MPR Fig. material with (supplementary colocalized TGN GFP–MPR 1 variant perinuclear PPAP2A The S3A,B). 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MOCK immunoprecipitated was these method were from control (SILAC) proteins culture phosphotyrosine-containing both states cell in labeling phosphorylation isotope to acids stable tyrosine amino the cells, by whose SRC-depleted in proteins modified were identify SRC. of upstream To suggesting functions receptor 5B,C), neurotrophin NTRK3 (Fig. the cells that TGFBR2-depleted not but NTRK3- aeilFg 4,) hnMC hshrlto was phosphorylation phosphatase MLC2 (protein PPP1R12A When down knocking fluorescence S4A,B). by mean augmented Fig. the supplementary 5E; increased material (Fig. also MLC2 NTRK3, Ser19-phosphorylated but of PAK3, or intensity or 2005). GIT2 TGFBR2 al., of et depletion SRC, that Zhang found 2009; we chains Accordingly, al., light et regulatory by its (Vicente-Manzanares activity of (MLC2) II state myosin phosphorylation controls the GIT1 regulating and complex PAK3 The GIT2, formation. containing carrier tubular GFP–MPR to coupled was q-value FDR oprbet hs bevdi el rae ihsi with treated cells in observed those to comparable material (supplementary of (siMYOIIA) depletion 3). chain upon tubules 1, Movies caused GFP–MPR heavy was of IIA phenotype number myosin similar the A in 5F,G). increase (Fig. an we phosphatase, observed MLC2 the also of subunit a 12A), subunit regulatory 1, asdtefamnaino h eiula GFP–MPR mimicking perinuclear thus the 6J), (Fig. of experiments) (91 By h fragmentation 1 6C), 6J). for (Fig. (Fig. compartment PD173074 the inhibitor TGN integrity FGFR the caused the Golgi with of at incubation affecting number As contrast, forming the significantly increased carriers cells. without h tubular 1 GFP–MPR-expressing for GFP–MPR SB431542 in inhibitor TGF- si signaling alter signaling above, to ligands mentioned FGFR and these inhibitors understand specific and better Different used trafficking To we MPR transport. S3). aspects, of Table aspects MPR material distinct (supplementary regulate and to appear dynamics of receptors dynamics cytoskeleton the transport. actin controls MPR that retrograde not pathway but a tubules, MPR to PAK3 TGN-derived belong containing complex GIT2 regulatory actin that and as suggest an TGN, data and These the SRC 6H,I). to NTRK3, (Fig. and GFP–MPR uptake endosomes affect anti-GFP to by not surface measured cell did the formation from genes tubule recycling the MPR of depletion increased indicated However, that colocalization. as of 6F,G), extent (Fig. their to compartment by and perinuclear 6D,E) 6B,C; the GFP– (Fig. (Fig. in ciMPR experiment, TGN46 endogenous data this to similarly In behaved siRNA S4C–G). MPR SRC Fig. the Golgi the material caused confirming which supplementary and inhibitor, thus kinase Rho AG879 the fragmentation), not inhibitor (but 1 TGF- neurotrophin inhibitor the was the with phenotype incubated SB431542, same cells 6A,C; The (Fig. in 2). microscopy observed 1, time-lapse Movies by material seen supplementary as 21), phosphatase PAK3 hs ysnI-eedn hne ntbl yaiswere dynamics tubule in changes myosin-II-dependent These u aaietf ikbtensgasfo h elsurface, cell the from signals between link a identify data Our ,si ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal NTRK3 5 .3,spotn h yohssta ci dynamics actin that hypothesis the supporting 5.33), ,si 6 4 ftecls mean cells; the of 14% TGFBR2 TGFBR2 ricbto ihteTGF- the with incubation or rsi or DUSP21 SRC tetdcls and cells, -treated b FGFR1 inln inhibitor signaling da specificity (dual 6 SRC knockdown s.d., ,si 2 GIT2 5085 log n 5 10 b b 3 ,

Journal of Cell Science EERHARTICLE RESEARCH 5086 ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal vr 0 sfrattlo i.Iae were Images min. 2 10 of bars: total Scale a inverted. for ms 500 every acquired were 1–3) Movies Images material microscopy. (supplementary time-lapse by analyzed were oto,sMOI n si and siMYOIIA MOCK control, (black) GFP–MPR-expressing loading (G) a control. as anti-GAPDH chain, and heavy anti-PPP1R12A anti-MYOIIA by using evaluated blotting were knockdowns western 3) and 2 si 1, and (siRNAs siMYOIIA of Efficiencies (F) § niae iNswr aee ihanti-phospho with the labeled with were treated siRNAs Cells indicated (E) dynamics. tubule MPR both ( in experiments abundance in were changed red significantly in marked Proteins mass analysis. for spectrometry processed and immunoprecipitated were proteins Tyrosine-phosphorylated cells. transfected e elwr esrduigImageJ using (median measured were intensities cell fluorescence per mean and MLC2 (Ser19) efre ihMC-rnfce n si and MOCK-transfected were with labeling performed reverse and SILAC forward Two with substrates. experiments SRC of analysis based ** quantified and (median (B) (C) blotting western were by lysates analyzed whole-cell in in and levels immunoprecipitates SRC anti-phosphotyrosine 4G10. the antibody with (pTyr) lysates cell from (IP) immunoprecipitated Tyrosine- were (B,C) proteins analysis. phosphorylated visual formation by tubule seen GFP–MPR as in increase depletion an which caused for red genes The indicate proteins. quadrilaterals regulatory signaling actin with and connected receptors hub central a SRC represents view). stronger confidence indicate (STRING lines associations and Thicker 1 4A). clusters (Fig. in found 2 genes the for analysis performed association was protein functional STRING (A) biogenesis. carrier control tubular proteins GFP–MPR regulatory Actin-myosin 5. Fig. P 0clsfrec odto;* condition; each for cells 50 , .0) D uniaiems spectrometry- mass Quantitative (D) 0.006). 6 vrg deviation; average 6 P vrg deviation; average , .) EG YIAcnrl GFP– controls MYOIIA (E–G) 0.1). PPP1R12A m m. n 5 P experiments; 3 , n § 0.025). PPP1R12A tetdcells -treated experiments; 4 SRC -

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Journal of Cell Science K eu-tre n nuae ih1 gm TGF- ng/ml 10 with incubated and serum-starved (K) 10 control, DMSO epciesRA,adte iha nioyaantGP(e)fr1hon h 1 for 37 (red) at GFP incubated against then antibody were an Cells with ice. the then with and incubated siRNAs, were respective cells GFP–MPR-expressing (H,I) condition). cl as 10 bars: ( Scale Images ImageJ microscopy. using confocal by analyzed analyzed were and (red) anti-GM130 with labeled ( h) (1 FGF2 experiments; i sRA n OK r1mn(MOadihbtr)(supplementary inhibitors) and (DMSO min 1 of or total a MOCK) for and ms (siRNAs 500 min every acquired 2 tubule were the Images and condition). per region experiments TGN (median the quantified in were Arrows forming lengths black. or tubules the in min conditions, GFP–MPR shown indicated 2 of all is (A) number For GFP–MPR of (C) and tubules. total GFP–MPR inverted a TGN-derived were for indicate Images ms min. 500 every 1 acquired (B) were Images cells. treated si and MOCK- (A) in 6. Fig. ARTICLE RESEARCH ahne l,21;Kzke l,21;Mra ta. 2011; al., et Moreau 2013; 2010; al., 5088 al., et et Kozik to Collinet 2010; 2012; transport al., al., post-Golgi et et or (Chia Farhan transport membrane plasma Golgi the to ER of endocytosis, view coiled-coil comprehensive more transport. a MPR (Rho-associated, At provide of complex]. regulation data the PAK3–GIT2 our ROCK1 the time, and same the 1) 1), (LIM kinase LIMK1 protein containing 2), kinase effector by small domain (CDC42 transport [CDC42SE2 MPR proteins AMP- the kinase, of in (protein subunit activated, [PRKAA2 catalytic complexes kinase a protein (CSNK2B), signaling AMP-activated subunit CK2 these a a identifying confirmed for We al., 2005). al., protein role et et cAMP-dependent (Wilkinson (Scott dynamics the actin sorting controls of CI-MPR subunits GSK3 of kinase, catalytic regulator two CK2, known kinase 2006), casein a the of is subunit a which – This kinases 2009). five al., identified et study (Adachi trafficking CI-MPR regulates by signaling mechanisms transport. reveals post-TGN and regulates signaling GFP–MPR maps cell by It which by trafficking. followed MPR (represented routes control pathways the phosphatases) signaling and kinases how 127 illustrates screen Our steps. DISCUSSION regulatory distinct affecting by possibly endosomes, TGF- and compartment (e.g. pathways Golgi signaling peripheral different b the of conclusion, size In affecting 6K). the (Fig. without increased slightly objects, h with 1–4 treatment GFP–MPR for contrast, FGF2 By ng/ml 6K). 5 (Fig. microscopy fluorescence 70 10 with in compared compartment GFP–MPR perinuclear ng/ml 10 with Treatment TGF- S3A,B). Table material (supplementary condition each (median for software calculated Volocity were the correlation coefficients using Pearson overlap indicate (E,G) and Arrows TGN. (Rr) microscopy. the coefficients confocal from by emerging analyzed tubules were GFP–MPR (red) (red) ciMPR TGN46 (D) with (F) co-labeled or cells GFP–MPR (D–G) 1–3). Movies material nooa oprmn eecluae (median in calculated localized peripheral were anti-GFP the compartment in of and endosomal amounts compartment) analyzed the GFP–MPR-positive were (perinuclear between TGN Images ratios the (I) the region. and TGN ImageJ, the using indicate Arrows uptake. GFP n GRsgaig oto P rnpr ewe h TGN the between transport MPR control signaling) FGFR and eea iN-ae cen aebe sdt study to used been have screens siRNA-based Several cell that shown has screen kinase-inhibitor-based recent A b AB F–P yaiswr nlzdb ielpemicroscopy time-lapse by analyzed were dynamics GFP–MPR (A,B) ,aTFRlgn,fr4hcue rgetto fthe of fragmentation a caused h 4 for ligand, TGFBR a 2, n a . 5 aayi uui),a ela ci regulatory actin as well as subunit)], catalytic 2 m 5clsprcniin.(,)Clswr J nuae with incubated (J) were Cells (J,K) condition). per cells 45 experiments; 3 b M. n h D4-idn rti kinase protein CDC42-binding the and m GB niio r10 or inhibitor TGFBR M TGFBR2 6 .%i oto el,a enb confocal by seen as cells, control in 0.8% tasetdo B MO rTGFBR-inhibitor- or DMSO- (B) or -transfected n . 5 6 0clsprcniin.Clswr fixed, were Cells condition). per cells 50 experiments; 3 einaslt deviation; absolute median 6 ˚ o h niae ie,t lo anti- allow to times, indicated the for C s.d.; n 5 m experiments; 3 GRihbtrfr1h or h, 1 for inhibitor FGFR M . 6 0 el e condition). per cells 100 6 vrg deviation; average b 1 ftecells, the of 11% 4h r5ng/ml 5 or h) (4 2 . n § 0clsper cells 20 3 a which , n 5 3 rdcino A rmpopaii cdadlysophosphatidic and acid phosphatidic the from regulate include DAG might of These that production trafficking. PPAP2A that phosphatase MPR screens Golgi-localized or control other the the specifically 2011) in thus 43 identified al., found might not we et phosphatases Importantly, and 2008). (Orvedahl kinases al., et autophagy with (Ganesan found viral melanogenesis was overlap studying subunit, No PPP2CB screens homeostasis. catalytic 1), TGN CDKN3 kinase control 2, probably (NIMA-related suppressor [e.g. phosphatase tumor NEK7 proteins (large (protein 3), LATS1 regulatory 7), inhibitor cycle tyrosine kinase cell kinase avian (fms-related and FLT4 (cyclin-dependent [fer (v-erb-b2 4)} kinase], FER, 2), ERBB2 tyrosine kinase homolog related) kinase), oncogene AXL (fps/fes viral {e.g. tyrosine leukemia kinases erythroblastic tyrosine receptor receptor included regulators. (AXL common group some latter share trafficking The VSVG and suggesting virus 2012), MPR stomatitis al., that vesicular et (Simpson genes monitoring secretion 29 screen (VSVG) and glycoprotein a 2010), al., in et S4), identified Farhan through were 2012; Golgi Table affect al., endocytosed to et shown are (Chia material were morphology MPRs genes 17 (supplementary that Another pits. endocytosis notion clathrin-coated 2010) the clathrin-dependent with affect al., consistent to screen, et our shown in (Collinet been identified genes have the Among 47 2012). al., et Simpson h rti iaeCfml,PKG(rti iaeC kinase (protein C PRKCG kinase family, (protein be of C PRKCH members to kinase two remains include which protein also of the They role studies. the further 2003), by al., established et (Smyth (LPA) acid ta. 00 n PI-4,5P and 2010) al., et eeomn fmliellrognss(TGF- or b organisms dynamics) multicellular actin adhesion, of (cell development homeostasis regulating cell of cascades aspects signaling key Several trafficking. MPR 2012; regulates al., pathway. et sorting (Hirst 2000) AP-1-dependent 2007; al., an al., al., et et or et Popoff Meyer 2007) 2012; (Arighi al., al., et et retromer-dependent McKenzie Wassmer 2004; a to al., et endosomes along be Lin early 2004; would possibly from screen MPRs TGN, our 2008). of 2006), the recycling al., al., the et routes with Waguri et different consistent 2002; slightly GFP–MPR al., Vicinanza follow et our MPR (Barbero that 2006; full-length possibility the Camilli, the and reporter exclude De cannot we and Although Paolo (Di ee euaigtemtbls fP-Po h og,P-Pon PI-3P TGN Golgi, PI-3,4,5P the the on endosomes, to PI-4P then of early metabolism and the regulating endosomes genes that Dı late 2012; suggest Hoflack, to and TGN, others that (Anitei transported the suggest and are to studies endosomes, back they Some recycling endosomes clear. through early yet recycling form perhaps not their transported are are allowing TGN, MPRs their those the membrane, to particular plasma back in the routes, and endosomes trafficking TGN, the endosomes. or between TGN 21 the of control on receptors present group these machineries how a and sorting whether the identify clarified be endosomes. we to and remains TGN Here, in between It transport involved known. MPR control GPCRs not that specific GPCRs are the process 2010), al., this et 2014; (Saini al., exceptions et protein (Coria G formation Dı specific carrier how post-TGN illustrated control subunits have studies Several GPCRs. (Dı transport Golgi ctnn ouaeMRtasoto h oesai fthe of homeostasis the or transport MPR modulate -catenin) ´ hssuypoie opeesv iwo o signaling how of view comprehensive a provides study This lhuhi swl cetdta Psaei equilibrium in are MPRs that accepted well is it Although An az ora fCl cec 21)17 0959 doi:10.1242/jcs.159608 5079–5092 127, (2014) Science Cell of Journal e n ahta 05.Nvrhls,wt some with Nevertheless, 2005). Malhotra, and ˜el ´ zAn az 2 e n ahta 05,adagopof group a and 2005), Malhotra, and ˜el n PI-3,4,5P and g 3 ´ ,wihi nw orglt post- regulate to known is which ), zadPefr 98.W identified We 1998). Pfeffer, and az nrccigedsms(Fields endosomes recycling on 3 ttepam membrane plasma the at b b inln,Wnt/ signaling, szm) that isozyme)] c and )

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Journal of Cell Science D-AEadwsenblotting. western and SDS-PAGE ufr eupne n20 in resuspended buffer, o IA xeiet,HL el eeclue nDE 45g/l (4.5 DMEM in cultured were cells HeLa experiments, SILAC culture For cell and labeling SILAC 4G10 or Sweden) for 4 CA) to at (Millipore, Healthcare, h Conjugate coupled 4 Agarose (GE (4G10) anti-phosphotyrosine Platinum antibodies beads anti-phosphotyrosine protein-A–Sepharose with incubated ARTICLE RESEARCH oshnseencat[rn ubr R 3121,H 5411 HO 2584/1-1, Deutsche HO from 83/1-2010, grants TRR by numbers part [grant in Forschungsgemeinschaft supported was research Our M.A. with Funding manuscript the B.H. wrote analysis. M.Z. and and and experiments, F.M. acquisition analyzed K.K., image and S.C., automated designed M.E., the M.B., screen, SILAC M.A. the the and performed analyzed S.L. data and with the performed together did designed, experiments R.C. C.C. experiments. M.A. the with analyzed analysis and performed designed, M.A. contributions Author interests. competing no declare authors The interests Competing and technical comments for critical Germany) and assistance. Dresden, discussions Universitaet helpful (Technische their Pursche for Theresia members lab thank We MTMR4–GFP (CNRS-Universite for Barnier UK) Lexington, University, Jean-Vianney Medicine, (Aston and Wassmer of Thomas College HA–PPAP2A, Kentucky for of KY) (University Morris Andrew thank We Acknowledgements (3 column trap nano an C18 on PepMap separated were a Peptides with Netherlands) 2006). The equipped Amsterdam, al., (Dionex, system nanoHPLC described et UltiMate3000 as performed (Czupalla were extraction previously peptide and digests tryptic In-gel spectrometry Mass L- adding media by SILAC ‘light’ prepared Arg-0/Lys-0 calf were and fetal ‘heavy’ dialyzed Arg-6/Lys-6 (v/v) (FCS). 10% serum with supplemented medium glucose)-based mn cd,rsetvl,adclswr utrdfr71 asin tyrosine-phosphorylated days 7–14 of above. for described as cultured Immunoprecipitation performed were was proteins cells medium. non-labeled and corresponding SILAC respectively, the or acids, MA) amino Andover, Laboratories, Isotope 2cm (3 aiu ftomse laaests etd dniiain were identifications Peptide until (4) probability sites. error posterior respectively; cleavage their on missed mass based oxidation, accepted cysteine ion two modifications, fragment of methionine mass the and variable maximum ion (2) and and and precursor fixed trypsin; entries for carbamidomethylation (3) observed Da all specificity, respectively; 0.5 frequently of enzyme and tolerance, ppm sequences (1) 255 6 reversed criteria: accuracy, and as search 2011_02) well following UniProt-KB/Swiss-Prot as the (release from contaminants database entries against 20,253 searched human were intense containing lists most Peak database eight 2008). MaxQuant a using Mann, were the 5–45% performed and of was (Cox spectra analysis scans 1.2.0.11 of Data version MS/MS Mass LTQ. the and (resolution gradient in nl/min. scan Orbitrap ions 200 survey precursor the linear MS in at one 60,000) with 80-min acid mode of data-dependent an formic a in 0.1% acquired with Germany). in Bremen, eluted acetonitrile Scientific, were Fisher (Thermo Peptides Orbitrap spectrometer LTQ a mass of XL Denmark) Odense, (Proxeon, source nanoelectrospray eertie,wiepoenfledsoeyrtswere rates discovery false protein while retained, were IA aiso tlattoppie e ape eut eeonly were median Results Protein was sample. the ratios per identified. protein on of peptides variation were , two based experiment-to-experiment least the peptides MaxQuant if at included two of by ratios least performed SILAC at was if quantification considered were 0 ntoidpneteprmnswt IA ae swapping. label SILAC with experiments independent two in 30% m ,10A 100 m, 6 75 ˚ ,wt oain ed eewse ietmswt lysis with times five washed were Beads rotation. with C, m nenldaee)adaPpa 1 nltclcolumn analytical C18 PepMap a and diameter) internal m ˚ ,15cm 6 75 m m nenldaee)drcl ope othe to coupled directly diameter) internal m 13 lof4 C 6 agnn n L- and -arginine 6 aml ufradaaye by analyzed and buffer Laemmli ai-u,Fac)frPAK3–GFP. for France) Paris-Sud, ´ 13 C 6 lsn (Cambridge -lysine , %rvrehits reverse 1% , % Proteins 1%. m ,10A 100 m, ˚ , ils .C,Kn,S . hen . ag .S n Fo and S. R. Kang, E., Shteyn, M., S. King, C., I. Fields, Dı Dı Farı R., Sharan, Y., Silberberg, E., Fava, S., Mitrovic, W., M. Wendeler, H., Farhan, P. Camilli, De and G. Paolo, Di B. Hoflack, and E. Krause, D., Thiel, T., Riedl, H., Mansukoski, C., Czupalla, M. Mann, and J. Cox, oi,A . asrn,M .adDı and L. M. Masseroni, S., A. Coria, dci . ao . ad,T .adMrt,M. Murata, and C. T. Saido, F., Kano, A., Adachi, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.159608/-/DC1 online available material Supplementary material Supplementary olnt . Sto C., Collinet, uddb uoenSrcua ud gatnme FE317 T135]. UT 301270 EFRE number [grant is Funds R.C. Structural TU-Dresden. and European 2584/9-1]; by HO funded and 2584/8-1 HO 2584/6-1, HO 2584/2-1, abr,P,Btoa .adPefr .R. S. Pfeffer, and L. Bittova, P., Barbero, B. Hoflack, and M. C., J. Anitei, MacMillan, W., M. Partington, S., Bagrodia, G., J. Gleeson, M., K. Allen, B. Hoflack, and U. Bauer, A., Alconada, ha . o,G,Rcn,V,N,S,Kmr .adBr,F. 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