Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/parameters.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/summary.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/proteinGroups.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/mzRange.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/modificationSpecificPeptides.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/OxidationMSites.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/msmsScans.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/allPeptides.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/evidence.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/msms.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/peptides.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031352_MQQC_CL_EMA_S06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031367_MQQC_CL_EMA_S21.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031371_MQQC_CL_EMA_S25.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031357_MQQC_CL_EMA_S11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031348_MQQC_CL_EMA_S02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031346_MQQC_CL_EMA_S01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031355_MQQC_CL_EMA_S09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031358_MQQC_CL_EMA_S12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031372_MQQC_CL_EMA_S26.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031350_MQQC_CL_EMA_S04.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031354_MQQC_CL_EMA_S08.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031362_MQQC_CL_EMA_S16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031364_MQQC_CL_EMA_S18.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031374_MQQC_CL_EMA_S28.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031363_MQQC_CL_EMA_S17.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031356_MQQC_CL_EMA_S10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031370_MQQC_CL_EMA_S24.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031349_MQQC_CL_EMA_S03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031368_MQQC_CL_EMA_S22.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031359_MQQC_CL_EMA_S13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031369_MQQC_CL_EMA_S23.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031360_MQQC_CL_EMA_S14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031376_MQQC_CL_EMA_S30.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031361_MQQC_CL_EMA_S15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031375_MQQC_CL_EMA_S29.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031351_MQQC_CL_EMA_S05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031365_MQQC_CL_EMA_S19.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031366_MQQC_CL_EMA_S20.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031353_MQQC_CL_EMA_S07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061234/Qplus_00031373_MQQC_CL_EMA_S27.rawprimary200OKgunnar.dittmar@lih.luEleanor MartinMass SpectrometryBottom-up proteomicsEpithelial polarityhttps://www.ebi.ac.uk/pride/archive/projects/PXD061234Epithelial CellMDCK-II cells stably expressing Pals1-A2E were incubated in media containing 2.5 mM biotin phenol (Iris Biotech) for 30 minutes at 37 °C. Cells were washed twice with PBS and the APEX reaction was initiated by incubating with 250 mM H2O2 for 1 minute at room temperature. The APEX2 reaction was quenched by washing three times with the quencher solution (PBS containing 5 mM (±)-6-hydroxy-2,5,7,8-tetramethylchromane- 2 carboxylic acid (Trolox), 10 mM sodium ascorbate, 10 mM sodium azide) prior to lysis in ice-cold lysis buffer (1 % sodium deoxycholate in 50 mM ammonium bicarbonate pH 8.0). centrifugation at 20,000 xg for 30 min at 4 °C. Protein concentrations were measured by Bradford and equalized. High-capacity streptavidin sepharose beads (GE healthcare) were washed once with 40 % methanol and twice with ice-cold lysis buffer (1 % sodium deoxycholate in 50 mM ammonium bicarbonate pH 8.0) prior to incubation with protein lysates (50 μL bead slurry per 3 mg total protein) for 2 h at 4 °C on a rotator. Beads were then washed twice with cold lysis buffer, and then once with each of the following washing buffers: 1 M KCl, 0.1 M Na2CO3, 2 M Urea in 50 mM ammonium bicarbonate pH 8.0. The last two washes were performed with cold 50 mM ammonium bicarbonate pH 8.0. Beads were then re-suspended in 2 M Urea in 50 mM ammonium bicarbonate pH 8.0 and protein reduction was performed by incubating with DTT at a final concentration of 10 mM for 45 min at 37 °C and 800 rpm. Samples were subsequently alkylated through incubation with iodoacetamide at a final concentration of 25 mM for 30 min at room temperature and 800 rpm. Samples were pre-digested with 0.5 μg LysC for 4 h at 37 °C and 800 rpm and then digested with 1 μg trypsin overnight at 37 °C and 800 rpm. Peptides were acidified to pH 2 by adding formic acid to 1 % final concentration and centrifuged for 15 min at 16,000 rpm at 4 °C to remove any precipitates or beads on Sep-Pak μ-C18 Elution Plates. Peptides were then dried on a speed vac for 2 hours and re-suspended in 0.1% formic acid prior to LC-MS. Scientific) LC system equipped with an Acclaim PepMap RSLC column (15 cm × 75 μm, C18, 2 μm, 100 Å, Thermo Fisher Scientific). Peptides were eluted at a flow rate of 300 nL/min in a linear gradient of 2-35% solvent B over 70 minutes (solvent A: 0.1% formic acid; B: 0.1% formic acid in acetonitrile (ACN)), followed by a 5 minutes washing step (90% solvent B) and a 10 minutes equilibration step (2% solvent B). Mass spectrometry was performed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray source and using uncoated SilicaTips (12 cm, 360 μm o.d., 20 μm i.d., 10 μm tip i.d.) for ionization, applying a 1500 V liquid junction voltage at 250°C capillary temperature. MS/MS analysis were performed in data dependent acquisition (DDA) mode, and the 12 most intense parent ions were fragmented with a resolving power of 17500 (at 200 m/z). Automatic gain control (AGC), maximum fill time and dynamic exclusion were set to 1e6, 60 ms and 30 ms respectively.PrideProtein quantification was performed with MaxQuant (version 1.6.17.0) using the following parameters: carbamidomethyl cysteine as fixed modification, methionine oxidation and N-acetylation as variable modifications, digestion mode trypsin specific with maximum 2 missed cleavages, and an initial mass tolerance of 4.5 ppm for precursor ions and 0.5 Da for fragment ions. All experiments were performed in quadruplicate except for the no peroxide sample which was performed in triplicate. The abundance of assembled proteins was determined using label-free quantification with the match between runs option was selected. Protein identification was performed searching against the Uniprot reference proteome (Uniprot ID 9615, downloaded on 06 January 2020).ProteomicsGunnar DittmarGroup Leader, Proteomics of Cellular Signaling, Luxembourg Institute of HealthPARTIALCanis Familiaris (dog) (canis Lupus Familiaris)eleanor.martin@ntu.edu.sgNot availableNTU (former)United KingdomfalseBulk delivery of a preassembled apical surface initiates epithelial lumen formationThe creation of a microvilli-rich apical luminal domain is essential for epithelial tissue morphogenesis and is closely coordinated with the formation of the apical junctional complex (AJC). De novo lumenogenesis, in which epithelial cells establish apical identity by directing apical cargo to an apical membrane initiation site (AMIS), has been studied extensively. However, the mechanisms behind AMIS formation and its progression into a luminal precursor remain poorly understood. In this study, we employed super-resolution imaging, correlative light and electron microscopy, and time-resolved proximity proteomics to explore the spatial, temporal, and molecular processes involved in apical lumen initiation in MDCK-II cells. Interestingly, we discovered that, in both 2D and 3D cultures, the formation of the apical cell cortex begins with the fusion of large intracellular apical precursor organelles called vacuolar apical compartments (VACs) at developing cell-cell junctions. Our time-resolved proteomics and imaging analyses revealed a dynamic protein network that likely coordinates the exocytosis of VACs and lumen initiation with the formation and maturation of the AJC. Furthermore, we report that loss of the Crumbs complex proteins Pals1 or PatJ or inhibition of Rho/ROCK signaling impedes VAC exocytosis at the AMIS, resulting in severe defects in lumen morphogenesis. Overall, our findings define a molecular network for de novo cell polarization and highlight Pals1/PatJ as key regulators of apical domain initiation. We further propose that VACs represent a previously unrecognized transport carrier that plays a crucial role in the rapid establishment of apical domain identity.2026-06-082025-02-26PXD061234NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:1316931NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:511145NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:243277NEWT:990119NEWT:7067NEWT:2850NEWT:1111708NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:272844NEWT:54571NEWT:9598NEWT:8030NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:1679NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:40479NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:7159NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:27448NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:40559NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:186990NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:49240NEWT:334747NEWT:61235NEWT:343691NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NEWT:206411NCBITaxon:6191NEWT:596153NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:52638NEWT:7004NEWT:4097NCBITaxon:6073NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:1329349NEWT:7370NEWT:83906NEWT:607699NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:208963NEWT:208964NEWT:27337NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:9935NEWT:89453NEWT:4120NEWT:51515NEWT:5693NEWT:2186NEWT:8724NEWT:51511NEWT:8845NEWT:92867NEWT:8723NEWT:92866NEWT:5334NEWT:37334NEWT:51750NEWT:382352NCBITaxon:10359NEWT:1203258NEWT:242619NEWT:160791NEWT:632957NEWT:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:89462NEWT:8839NEWT:4232NEWT:2758385NEWT:37349NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:312017NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:375939NEWT:61674NEWT:870435NEWT:1117957NEWT:6526NEWT:9913NEWT:8709NEWT:105841NEWT:337330NEWT:4100NEWT:2666255NEWT:1159556NEWT:5671NEWT:2285NEWT:1076NEWT:4101NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:1486917NEWT:981087NEWT:180066NEWT:226900NEWT:468911NEWT:817NEWT:7604NEWT:818NEWT:135588NEWT:1843183NEWT:5661NEWT:563041NEWT:29719NEWT:58002NEWT:326423NEWT:452467NEWT:36111NEWT:326424NEWT:6997NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:749907NEWT:1042986NEWT:1911079NEWT:69373NEWT:145263NEWT:264203NEWT:1480154NEWT:694569NEWT:1274414NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:9612NEWT:38626NEWT:38865NEWT:51953NEWT:3077NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:112273NEWT:36329NEWT:1147787NEWT:62615NCBITaxon:3044782NEWT:1440772NEWT:72407NEWT:1355477NEWT:349741NEWT:9605NEWT:9606NEWT:90371NEWT:157295NEWT:641501NEWT:4153NEWT:2096NEWT:7668NEWT:915099NEWT:74940NEWT:13551NEWT:9721NEWT:137221NEWT:9600NEWT:9963NEWT:1450511NEWT:1143193NEWT:1617910NEWT:174934NEWT:565050NEWT:2823525NEWT:2785785NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:9839NEWT:58334NEWT:1193501NEWT:3055NEWT:58331NEWT:4146NEWT:5478NEWT:6326NEWT:6689NEWT:5476NEWT:1450520NEWT:299767NEWT:28835NEWT:1274432NEWT:2053667NEWT:1274426NEWT:70448NEWT:9825NEWT:1274423NEWT:393765NEWT:698936NEWT:39946NEWT:9823NEWT:9940NEWT:95602NEWT:8732NEWT:521001NEWT:192011NEWT:1813887NEWT:39947NEWT:224911NEWT:1274420NEWT:5784580000-0003-4707-697X