Journal of Cell Science el Gnesne l,18) nnuolsoaclsdetyr-tubulin cells TC-7 neuroblastoma from In 1984). microtubules al., interphase metaphase et of (Gundersen subset the cells been limited and only a are have in network microtubules microtubules detected detyrosinated interphase stable tyrosinated the whereas Dynamic spindle, in division. found has different during detyr-tubulin cell commonly and and types of tyr- cell different of phases in distribution intensively essential The studied is to 2005). been on due enzyme al., birth after this et hour acts that (Erck 1 within suggesting 1975). mice disorganization, in which Flavin, lethal neuronal is and TTL (Raybin (TTL), of of knockout tyr-tubulin A synthesis restores ligase the and on heterodimers tyrosine dependent is kinesin tubulin tyrosination centromere-associated by Tubulin modulated mitotic be (MCAK). motor can process depolymerizing microtubule This 2009). of the al., inhibition et and (Peris stabilisation disassembly to leads C-terminus detyrosination the to tyrosine of of re-ligation cycle subsequent a and and by removal (tyr-tubulin) of generated modification tyrosinated are of 2008). microtubules tubulin-specific Pools (detyr-tubulin) 1973). al., detyrosinated first al., et the et (Barra reported was (Hammond tyrosination phosphorylation to Tubulin subject and is palmitoylation Tubulin 1995). detyrosination, acetylation, al., integrity include tyrosination, et which the modifications, (Yap post-translational affect sheets distinct (Mu thus cell pole epithelial composition the cell of microtubule basolateral in transport complete the in roles specific or Alterations apical and critical a the polarity play to apico-basolateral pathways and cytoskeleton, an as the of Microtubules, domains generation of architecture. elements membrane cytoskeletal central the two of reorganization into plasma the membrane of separation the involves cells epithelial of Polarization Introduction suggests cells. which MDCK knockdown, polarized TTL in by cargo reversed membrane the be guide words: could for to to Key effect required particular tracks This in cytoskeletal is cells. trafficking, as detyrosination TTL-overexpressing membrane serve tubulin in Moreover, microtubules down Thus, monolayer. detyr-tubulin-enriched cells slowed architecture. epithelial that was Detyr-tubulin-depleted an polarized domain, well cell establishment. into prematurely apical levels monolayer cells MDCK not a the the non-polarized disrupted of developed decreased is from (TTL), which formation and differentiation epithelium ligase tyrosination, islands morphological the tubulin tyrosine polarized of isolated tubules in tubulin induction These into a of tubulin Constant sheets. assembled tyr-tubulin. overexpression of cell (tyr-) and confluent by detyr- tyrosinated maintenance into detyr-tubulin of assembly and of and stretches during alternating (detyr-) detected development of were detyrosinated composed microtubules the were detyrosinated between of in balance quantities Increased modifications the monolayers. tubulin studied We post-translational understood. of role The Summary 10.1242/jcs.109470 doi: 5998–6008 125, ß Science Cell of Journal 2012 October 5 Accepted Philipps-Universita ( Pathology, correspondence Cell for Ba and *Author Sebastian Biology Cell Freikamp, of Andrea Department Grosse, cells Lena Zink, epithelial Sabrina in trafficking apical formation and monolayer promotes detyrosination Tubulin 5998 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. a tbln hra yai irtblsaetyrosinated, are microtubules dynamic Whereas -tubulin. uui,Aia rnpr,Cl oaiy ottasainlmodification Post-translational polarity, Cell transport, Apical Tubulin, [email protected] D oiiain polyglutamylation, modification, 2 ) sh 2004). ¨sch, ab -tubulin tMrug abr,Germany Marburg, Marburg, ¨t hthv salse elcl otcsta nioae el day 1 cells (Bre isolated cells in in seeding than higher is contacts after tyr-tubulin cell-cell to established detyr- have of that ratio a Weber, the and with general, (Wehland processes, bodies In cell 1987). cell the in elongated tyr-tubulin of of concentration microtubules to restricted was qal itiue ln h pclbsltrlai.Steeper axis. apical–basolateral were gradual the tyr-tubulin a and along Detyr- by pole. distributed by followed cell membrane, equally tubulin basal studied three the apical towards all the first cysts decline of to accumulation cell was close an MDCK variants showed of cells Matrigel in analysis epithelial grown scan Line polarized immunofluorescence. tyrosinated in and acetylated tubulin detyrosinated, of arrangement The in cells tubulin epithelial tyrosinated polarized and detyrosinated of Distribution Results fully in of trafficking assembly protein correct cells. apical the polarized guiding cells; and MDCK which monolayers of into processes involved affected, cells two are least were at microtubules pathways in detyr-tubulin-containing transport that apical indicates particular molecules. cargo Constant in membrane-bound In the and resulted secreted perturbed cells. of and TTL delivery formation, surface of monolayer MDCK for overexpression detyrosinated periods time by of of prolonged detyr-tubulin number polarization of the depletion in during decrease tyrosination a microtubules an by found to to We followed sheets. detyrosination us cell increase epithelial tubulin prompted polarized of formation This of the during transport. dynamics kinesin-1 the Tubulin of motor by 2009). examine polarity regulation navigate Setou, cellular precise determine and to to a potential (Konishi cue the axons has directional thus to modification a kinesin-1 of as heads acts tubulin tyrosinated ne,Fak Mu Frauke ¨nfer, ´ ta. 91.I oaie ernlcells neuronal polarized In 1991). al., et shadRl Jacob* Ralf and ksch ¨ eerhArticle Research a - Journal of Cell Science hc eet loecn ysi ls rxmt oteaia or apical the to an proximity close microscopy, in TIRF showed dyes by and studied fluorescent detyr- was detects (Jaulin of who poles which quantity pole cell the two cell study, Kreitzer the which at further apical tyr-tubulin a S1), the and In 2010). at Kreitzer, Fig. tubulin Jaulin and acetylated material tubulin from of supplementary enrichment acetylated data also for supports see observed 1A; occasionally (Fig. were gradients y-o nidtrtblnatbde.A eitdi i.1 short 1, Fig. in depicted As on antibodies. seeding anti-detyr-tubulin after or anti- day with tyr- 1 immunostained then for methanol, grown with fixed were and coverslips cells The microscopy cells. (GSDIM) MDCK state return of ground molecular high-resolution individual with by depletion data analysed then scan was line which microtubules the confirms 1B), (Fig. and cysts. cells domain cell polarised MDCK of concentrates from basal poles preferentially and the variant were tubulin at tyr-tubulin apical neither and that the detyr- indicates Fig. of at material ratios detected (supplementary double similar tyr-tubulin was Overall, and proportion S2A,B). smaller detyr- a by while stained were (Schneider tyr-tubulin, microtubules cells membrane-proximal in the MDCK enriched of Most polarized 2010). of al., et membrane plasma basal h itiuino ey-adtrtblno double-positive on tyr-tubulin and detyr- of distribution The pteilitgiyaddtrtblndpein5999 depletion detyr-tubulin and integrity Epithelial ln irtbls(upeetr aeilFg S2C). Fig. using material (supplementary microtubules microtubules along Similar directed metaphase antibodies However, ends. and 1986). against al., preferential interphase tubulus et (Geuens for a immuno-EM had the stretches seen with tyr-tubulin at and been variants detyr- interspersed tyr-tubulin tubulin of arrangements string-of-pearls-like two of a the to concentration leading of thus appearance tyr-tubulin, of segments 1 to up of sections detyr-tubulin aeilFg 2) estmti uniiaino the of quantification Densitometric (supplementary tubulin S2E). acetylated or after Fig. detyr- harvested material tyr-, lysates by against and directed confirmed cell antibodies tubulin with was MDCK intervals incubation This of different S2D). analysis Fig. immunoblot polarisation material detyr- cell epithelial that polarisation. of (supplementary process suggested analysed the epithelial data during and decreased Immunofluorescence filters tubulin of days. on 1–5 incubated course were after the cells tubulin MDCK during two Therefore, the between varies balance the variants whether addressed we Next epithelial during polarisation tubulin detyrosinated in Fluctuations a tblna oto eemr rls vnydistributed evenly less or more were control a as -tubulin ey-adtrtbln epciey ncls1dyafter day 5 1 bars: label cells Scale to on seeding. used respectively, were tyr-tubulin, 647 and Fluor detyr- Alexa and 488 Fluor Alexa AD oto.Sgiiac a etdwt Student’s with tested t was a Significance to control. normalized GAPDH were anti- antibodies and tubulin anti-detyr- acetylated anti-tyr-, intensities anti-tubulin, band with in The detected depicted S2D. intervals Fig. time material indicated supplementary the after lysed cell ( Volocity ( the package using software in S2A,B images Fig. TIRF material from supplementary tyr-tubulin and detyr- ( of S1A,B). in Fig. indicated material (line supplementary axis apico-basolateral the along tubulin cells. MDCK in ( modifications Tubulin 1. Fig. ts (*** -test D A uniiaino h muoltdt rmMDCK from data immunoblot the of Quantification ) acetylated or detyr- and tyr- of analysis Linescan ) P , 0.0001), m n ogwr irpe ylonger by disrupted were long m 5 ) ( 4). m n 5 ;elre etos 1 sections, enlarged m; 7. C o SI microscopy, GSDIM For ) B Quantification ) m m. a - Journal of Cell Science omlzdt AD oto.Sgiiac a etdwt Student’s with tested was Significance (* control. GAPDH a to normalized h niae ubro asi utr o muoltigwt A anti- pAb with immunoblotting for culture in ( days TTL. of number indicated the pb niTLAeaFur56frcnoa irsoyo ( or microscopy confocal for 546 Fluor anti-TTL/Alexa (pAb) cells. MDCK in ( expression TTL 2. Fig. (MDCK TTL– cells (TTL–GFP). TTL MDCK GFP-tagged GFP-expressing of the expression enhance continuous to expected is TTL of increase tyrosination. tubulin an polarization since cell 1D), during accordance detyr-tubulin (Fig. in of is decline This observed polarized. quantities the fully with maximum TTL are a cells at 5, MDCK is day TTL when of level at synthesis the formed Hence, declined. been again once had monolayer 4. to cellular the 2 day low, Once from with polarization was cell changed during expression increased dramatically TTL expression but seeding TTL following Just 2B,C, As differentiation. Fig. control. loading A in a antibodies. as further depicted used anti-TTL and was with GAPDH electrophoresis analysis protein, housekeeping blot gel western cell SDS from by by protein processed on separated of expression amounts harvested were Equal TTL lysates were seeding. cells after in days MDCK changes different TTL-specific polarization, monitor epithelial with To during 2A). staining (Fig. immunofluorescence antibodies as by cells MDCK polarized of assessed cytosol the in into expressed detyr- is converts tyr-tubulin, that enzyme ligase the (TTL), tyrosine ligase tyrosine tubulin Tubulin detyr- by of detyr-tubulin pool of Alteration minor a detyr-tubulin, and of cells. tyr- polarised amount of fully prevalence the in tubulin high in initial a decrease an in a with resulting polarisation by modifications epithelial followed tubulin of increase of course pattern the the during Thus, changes 2011). al. et al., Quinones is et by decrease data (Quinones published immunofluorescence This previously 5. as our well day as corroborates at observations, 10% and than significant less to statistically day, decrease second a the as in by on maximum followed well is a to cellular which as increased of however, 1D), level detyr-tubulin, tyr-tubulin (Fig. overall the polarisation in with cell agreement increase during slow tubulin acetylated a shows immunoblots 6000 tbyepesn T–F eevrfe ywsenbo with blot western by verified were clones positive TTL–GFP and expressing neomycin of stably presence the in transfection after A P ie 5dy fe edn)adimnsandwt oylnlantibody polyclonal with immunostained and seeding) after days (5 fixed ) enx riiilydcesddtrtblni DKclsby cells MDCK in detyr-tubulin decreased artificially next We , .5 ** 0.05, C uniiaino h aadpce nB h aditniiswere intensities band The B. in depicted data the of Quantification ) ora fCl cec 2 (24) 125 Science Cell of Journal P , 0.007), n 5 .Saebr 10 bar: Scale 4. itrgonMC el were cells MDCK grown Filter m m. TTL–GFP a tbln h ee of level The -tubulin. eeselected were ) B ye after lysed ) t -test uuisadpeette rmbigicroae into incorporated being 2011). from al., tubulin et (Szyk them sequester described thus previously polarization prevent as may microtubules, cell and levels following TTL subunits Elevated decline 2B,C). normally (Fig. would enzyme ooaesi rae eal led y1dyatrplating, after day 1 by Already detail. MDCK greater in monolayers 3) h muto ey-uui a rmtclydcesdin decreased dramatically was MDCK Fig. detyr-tubulin of material amount (supplementary The antibodies S3A). anti-GFP and anti-TTL bu 0 fplrzdMC el eepstv o detyr- for positive were cells MDCK polarized of nocodazole, with 40% treatment hour slowly about 1 that a Following mM) S3C). (supplementary Fig. (10 material microtubules concentration detyr-tubulin a stable at was depolymerizes times Nocodazole nocodazole various 1988). a for al., added used et we (Khawaja detyr-tubulin, assay on depolymerisation effect the overexpression of assessment TTL quantitative of a For cells. MDCK control seen in be could tubules detyr-tubulin-enriched several whereas cells, hnsuidtefraino oaie MDCK polarized we of sheets, cell formation epithelial are the of of polarization studied formation level epithelial correct the then of the in for process changes required that the hypothesis during working detyr-tubulin our on Based TTL-overexpressing cells of MDCK morphology the in Changes polar detyr-tubulin-positive of number the MDCK contrast, In tubulin. ey-uui a aeydtcal nplrzdMDCK polarized comparison, Furthermore, in cells. detectable control direct barely to was compared detyr-tubulin In frequent 3C; less (Fig. were S3B). seeding cells TTL-overexpressing non-polarized after Fig. of microtubules days detyrosinated 1–4 material directed detyr-tubulin supplementary antibodies or tyr- using against microscopy immunofluorescence by ycntnl lvtdTLlvl nMDCK in of levels amount TTL explained the be elevated in can constantly balance decrease the changed by in a change This This with tubulin. S4). polymerized polarization, Fig. cell material following (supplementary seeding after infcnl lee.Terdcini ey-uui in not detyr-tubulin was in tubulin reduction acetylated or The tyr- MDCK altered. overexpression. of TTL significantly by concentration 50% about to The depleted also was tubulin aaidct htti olo tbemcouue a almost was microtubules stable of Our pool MDCK nocodazole. in this absent of that presence indicate the harbour data in detyr-tubulin-positive cells depolymerize slowly few MDCK which microtubules, a detyr-tubulin-enriched polarized stable of only Thus, amounts significant remained. time, cells of MDCK periods longer for nioisaanttr n ey-uui nplrzdepithelial MDCK polarized and in MDCK of detyr-tubulin mixture A and cells. tyr- against antibodies fasmldvru oul uui a loteulor equal almost was tubulin MDCK amount soluble in the elevated to versus Surprisingly, and significantly control supernatants. fractions assembled microtubule- a the pellet of as in the the material used in and Antibodies soluble where microtubules KIF5B between centrifugation. GAPDH discriminate protein by cytosolic motor associated dimers against tubulin microtubules directed soluble assembled assembled separated of of from therefore, level amounts total the non- We the in to influence microtubules. changes also so preferentially might 1978), enzyme binds al., this had et TTL It (Arce dimer. that tubulin soluble assembled shown to polymer been tubulin of previously ratio the affects cells eas drse hte vrxrsino T nMDCK in TTL of overexpression whether addressed also We TTL–GFP TTL–GFP TTL–GFP TTL–GFP el a eiidb muoitceitywith immunohistochemistry by verified was cells el hwdamc ihrtnec oji into join to tendency higher much a showed cells el a elgbe fe ooaoetreatment nocodazole After negligible. was cells TTL–GFP Fg AB.Itrsigy polyglutamylated Interestingly, 3A,B). (Fig. el ttesato h experiment. the of start the at cells TTL–GFP TTL–GFP el putl3days 3 until up cells el a analysed was cells TTL–GFP TTL–GFP el;the cells; TTL–GFP cell Journal of Cell Science ellyr3 or fe edn hra MDCK whereas seeding after hours 32 layer cell ahrnwr lge ale ln aea ebae in membranes lateral along earlier aligned MDCK E- were and ZO1 Moreover, cadherin vesicles. intracellular in still was claudin-1 oprsno h rudaeso h w ellnsrvae that MDCK revealed each lines plating, cell after two hours the of 12 areas ground S5B). the be Fig. material of could supplementary Comparison markers panel; lower polarity 4B, these no (Fig. of immunostained, detected distribution were days the 5 in after differences polarized cells If S5A). Fig. MDCK in contrast, In panel). eaetgt(upeetr aeilFg 5) E ausfor values TER S5D). MDCK Fig. material (supplementary tight became sebeit sad fpeaueyplrzdcls as cells, supplementary polarized to 4C; (Fig. tendency prematurely TTL MDCK analysis 2). 1, higher of Movies 4A). time-lapse material (Fig. a by islands scans demonstrated with x/z into correlated by assemble during indicated also taller become as overexpression cells formation the that island suggesting thus S5A,C), Fig. TR.Ti au nrae uigmnlyrfrainof formation monolayer to during used increased resistance be value MDCK lacunae transepithelial can This the extensive that is (TER). exhibited parameter formation still monolayer Another determine density islands. same cell between the at seeded 2husatrpaigi MDCK in assembled plating been after already hours had 12 junctions aligned laterally tight of membranes, part upper plasma by the indicated As in 4A). claudin-1 (Fig. of cells immunostaining MDCK than cells 10–30 of islands hs aasgetta T vrxrsini DKcells MDCK in formation. overexpression monolayer of TTL course the that in changes induces suggest summary, In data formation. monolayer these in delay a indicating controls, mle hna vrg MDCK average an than smaller TTL–GFP GFP TTL–GFP el ni trahdapaeuwe h monolayer the when plateau a reached it until cells el nrae lgtymr lwycmae to compared slowly more slightly increased cells hni MDCK in than GFP GFP GFP GFP el omda lotconfluent almost an formed cells TTL–GFP el splmnaymaterial (supplementary cells TTL–GFP oto el h aoiyof majority the cells control el(upeetr material (supplementary cell el Fg B upper 4B, (Fig. cells elwssignificantly was cell TTL–GFP cells pteilitgiyaddtrtblndpein6001 depletion detyr-tubulin and integrity Epithelial eeldsrn nrclua muotiigo p0in gp80 of immunostaining microscopy cells intracellular Confocal TTL-overexpressing MDCK S6A,B). strong in Fig. revealed affected material E-cadherin significantly and (supplementary membrane CD29 not proteins and basolateral was pronounced two a less the of with was trafficking trafficking cells Student’s basolateral TTL-overexpressing to on in according kinetics 0.01 secretion transport below of the significance gp80 in Quantification decrease apical substantial 5B,C). of a (Fig. revealed incubation data the of periods different eltdi MDCK in depleted pteilcls oti n,sraemlclso h pclor apical polarized the of MDCK in molecules of molecules surface membrane end, cargo this basolateral also of TTL To of transport cells. levels epithelial directed enhanced whether the study affect to wished cells we MDCK Next, of in delivery p75 surface and apical gp80 the modulates expression TTL rmnn rtisfo h pclad oalse xet from MDCK extent, stained. of lesser a silver surface to and, basolateral and apical the analysis the from SDS-PAGE proteins the cell Prominent by corresponding from the isolated separated to proteins comparison lysates of in streptavidin–Sepharose pattern domains membrane the with two depicts harvested 5A Fig. and beads. biotinylated were itra-iesrcue eedtce nMDCK in detected were structures cisternae-like lcpoenit h pclo aoaea eimfrom medium basolateral or sorted apically apical predominantly the this MDCK an of into Secretion (Wada or cells of glycoprotein 1994). MDCK delivery surface, of surface al., protein of cell secretory et cells. kinetics major the the depleted the gp80, studied detyr-tubulin soluble we to in this, cell trafficking elucidate the To forward into uptake in accelerated defect a TTL–GFP GFP MDCK ( (polyglut) polyglutamylated or acetylated by tyr-, analysed against were directed antibodies confluency with post immunoblot days 3 for incubated eenraie oGPH *** GAPDH. to normalized were niae nbu.Saebr:10 bars: are Scale Nuclei blue. 546). in immunostained Fluor indicated were Alexa tyr-tubulin (red, or antibodies Detyr- specific days. with 4 to 1 after fixed i.3 T vrxrsindpee detyrosinated depletes microtubules. overexpression TTL 3. 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TTL–GFP C oto cells control TTL–GFP )MDCKand cells cells Journal of Cell Science 6–) ncmiainwt h eut rmTTL-overexpressing MDCK from results the with Fig. E-cadherin combination material In (supplementary or S6C–E). knockdown CD29 TTL by of affected delivery not Basolateral was 6A,B). within (Fig. 25% by hours increased 4 to cells TTL-depleted gp80 of of (supplementary medium kinetics apical depletion the transport form Concurrently, TTL cells S7C,D). Fig. after MDCK material monolayers that revealed cell ZO-1 polarized and claudin control. markers E-cadherin, polarity Fig. mock the material of to (supplementary analysis microscopic depleted controls a Fluorescence S7A,B). to was compared as TTL 35% hours than transfection 48 less at siRNA transfection, siRNA luciferase Following and MDCK transfected cells transiently be in siRNAs TTL two would with levels performed in effect increase an opposite to The leading detyr-tubulin. depletion, of secretion. TTL upon protein reduction predicted dramatic a apical to leads detyr-tubulin in of level reduced a that Fg D,wihwr osbyedsmlognle,as MDCK in affected organelles, predominantly the are that pathways with suggest transport endosomal Together apical would this 1994). data, possibly al., biotinylation et surface were (Apodaca described previously which 5D), (Fig. 6002 xrsinsbtnilyatrteaia rnpr ieisof kinetics transport apical the gp80. soluble alter substantially expression hrfr,aseii iN-eitdkokono T was TTL of knockdown siRNA-mediated specific a Therefore, TTL–GFP ora fCl cec 2 (24) 125 Science Cell of Journal el,teedt niaeta hne nTTL in changes that indicate data these cells, TTL–GFP el and cells osblt,w nlsdsraedlvr fendogenously this of clarify To MDCK delivery the in 2003). surface to gp135/podocalyxin al., raft-associated due analysed in et expressed be step we (Jacob rate-limiting could pathway possibility, a but as transport microfilaments unexpected, this actin was of involvement no This observed be as could 6D). depletion, SI apical (Fig. TTL of quantities by the in affected changes significant not reduced apical was Lipid-raft-dependent with cells. however, the cells control trafficking, in reached in than higher had TTL 30% of that 6C amounts was p75–GFP Fig. membrane of transfection. plasma proportion apical siRNA the luciferase that or indicates depletion TTL after ncdw fedgnu T n TTL simultaneous and by effect TTL endogenous this for of compensation knockdown partial overexpression A TTL 6E). by (Fig. reduced detyrosinated significantly was of surface- of gp135 amount levels resident the biotinylation, depleted by assessed radically As microtubules. contain which cells, ht lofrarf-soitdgyorti,aia rfikn can trafficking apical glycoprotein, raft-associated a for also that, isnhtclylble n muorcpttdfo h two MDCK the polarized of from domains immunoprecipitated membrane and were 2007) labelled al., biosynthetically et sucrase (Delacour apical p75–GFP non-raft Lipid-raft-associated distinct and use (SI) pathways. isomaltase that and we polypeptides platforms cells, two transport TTL-depleted of delivery in the affected studied is glycoproteins associated ofrhrass hte pcltafcigo membrane- of trafficking apical whether assess further To eitd F loecnei niae ngen uliaeindicated are Nuclei green. ( in blue. indicated in is fluorescence GFP bars: depicted. Scale 546). Fluor (Alexa 10 anti-Claudin pAb with immunostained fe ,1 n 2husaedpce o ahcl ie(e also (see line 100 cell bars: each Scale for 2). depicted 1, are Movies hours material 32 supplementary and 16 0, after n MDCK and MDCK seeding, Scale antibodies. anti-tubulin 20 or with bars: 1 immunostaining for for fixed incubated and coverslips, days on 5 densities identical at of seeded islands were cells into cells. assembly MDCK the polarised promotes prematurely overexpression TTL 4. Fig. m .Freeytm on n tanx-cn n zsasare xz-scans and xy-scans strain and point time every For m. m C .( m. ielpercrigb I irsoyfo MDCK from microscopy DIC by recording Time-lapse ) TTL-GFP B 2hus(pe ae)o as(oe ae)after panel) (lower days 6 or panel) (upper hours 12 ) GFP el tre or fe edn.Iae obtained Images seeding. after hours 2 started cells n MDCK and TTL–GFP ( A el eefxdand fixed were cells SI DKadMDCK and MDCK ) –GFP rMDCK or Fg F suggests 6F) (Fig. m p75–GFP m. TTL–GFP TTL–GFP cells GFP Journal of Cell Science mgso muotie p0i oaie MDCK polarized in gp80 immunostained of images infcnewstse ihStudent’s with with immunoblotting tested by was analysed Significance and minutes ( 240 antibodies. or anti-gp80 120 5, after harvested seik ( asterisk. atrso h yae r eitdo h ih.Dpee rti candidates protein Depleted MDCK right. of the domains on surface depicted the Protein are from stained. lysates with silver the isolated and of was SDS-PAGE patterns material by biotinylated separated lysis beads, cell neutravidin Following cells. the of ( days. 5 for T eutdi usata nraei ey-uui,weesthe whereas of detyr-tubulin, for in Knockdown used increase cells. substantial were a TTL-depleted tubulin in tubulin resulted and acetylated TTL the control or of tyr- on immunoblots detyr-, antibodies first, knockdown against At 7). directed TTL (Fig. immunofluorescence and of immunoblots MDCK influence of composition the cells MDCK Finally, in knockdown TTL by detyr-tubulin of Elevation artificially not is TTL further of be level increased. the cannot if knockdown trafficking was TTL apical by that TTL optimized raft-mediated conclude we if of Thus, kinetics improved cells. apical TTL. the wild-type significantly SI, MDCK overexpressed not in for down was observed of knocked gp135 data level of the the delivery with conjunction lowering in by Moreover, 10 modulated bars: Scale blue. be in indicated are xz-scans and xy- in Nuclei cells. 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TTL without apical and the with to cells MDCK transported wild-type were MDCK gp135 in of surface amounts High gp135. protein marker ( n T–F fe iN transfection. siRNA after TTL–GFP and etrdtruhTLk.( TTL-kd. through restored irpe ydtrtblndpein hc a ersudby microtubules rescued detyrosinated that be suggests can This which knockdown. depletion, TTL is detyr-tubulin gp135 by lipid-raft-associated In or membrane. disrupted gp80 apical of the transport to apical non-raft-p75–GFP contrast, or gp80 soluble detyr- of of level cells. the MDCK in raised microtubules depletion in TTL tubulin ligase that the data these of demonstrate Altogether, levels S7E,F). clearly by Fig. normal material is expressing supplementary cells staining also cells (see MDCK detyr-tubulin in than 7D TTL-depleted stronger Fig. much in and seen control As immunofluorescence. in tyr-tubulin and altered detyr- monitored significantly of levels not were seeding, after were days tubulin Four 7A–C). acetylated (Fig. or tyr- of levels ih[ with iN cnrl a avse fe ,10o 4 iue n nlsdby analysed and minutes 240 ( or 120 5, ( after immunoblotting. harvested luciferase or was (TTL-kd) (control) siRNA siRNA TTL with transfected apical cells the MDCK filter-grown to efficiency transport domain. enhances membrane knockdown TTL 6. Fig. uniiain infcnewstse ihStudent’s and with analysis tested phosphoimager was by Significance followed quantification. immunoprecipitates SDS-PAGE, The to antibodies. surface subjected (b) corresponding were basolateral the or with (a) surface apical performed the Cell was from control. SI a or p75 as of siRNA immunoprecipitation luciferase or siRNA TTL E C ufc itnlto olwdb etr ltigaantteapical the against blotting western by followed biotinylation Surface ) n MDCK and ) nsmay lvtddtrtblnicesstetasotrate transport the increases detyr-tubulin elevated summary, In 35 TTL–GFP ]ehoiefr1 iue p5 r2hus(I ntepeec of presence the in (SI) hours 2 or (p75) minutes 15 for S]methionine SI el,tasoti evl mardadcudb partially be could and impaired heavily is transport cells, ( B D uniiaino he xeiet si .MDCK A. in as experiments three of Quantification ) el eegono itr n isnhtclylabelled biosynthetically and filters on grown were cells ) ( A eimfo h pclo aoaea oanof domain basolateral or apical the from Medium ) F nc onefcece fedgnu TTL endogenous of efficiencies down Knock ) t ts ( -test P , 0.05), n 5 p75 3. Journal of Cell Science rnfce ihlcfrs iN rTLsRAwr muotie gis ey-adtrtbln ncdw fiinyo T a sesdby 50 assessed bars: was Scale TTL control. of loading efficiency as Knockdown labelled tyr-tubulin. was and E-cadherin detyr- panel. against right immunostained the were in siRNA indicated TTL as or immunoblotting siRNA luciferase with transfected ihteidctdsRA n nuae o aswr nlsdb muoltigwt nioisaantTL uui,dtr,tr n acetylated and tyr- detyr-, tubulin, TTL, against antibodies with immunoblotting by analysed were days MDCK 4 from for immunoblots incubated The and siRNAs indicated the with nnreclshv ecie rdmnn oaino tyr- their of at whereas found bodies; location were cell microtubules in predominant of and axon domains a the detyrosinated of highly region described distal the have in tubulin variants cells on tubulin studies nerve cell modified previous in in post-translationally population contrast, poles of In each cell distribution basolateral monolayers. of the the epithelial amounts and or equal apical cysts the as at cells, detected these were was an in detyr-tubulin or of found tyr- domains of of not architecture distribution membrane polarized cell both A consequences to the cells. glycoproteins MDCK on of the variants transport tubulin the monitored and two these and in imbalance poorly detyr-tubulin only and is cells generation epithelial the polarized in understood. of modifications maintenance post-translational and of role The apical Discussion -independent cells. and MDCK lipid-raft-dependent in trafficking in protein involved are MDCK in microtubules detyrosinated elevates knockdown TTL 7. Fig. 6004 nti td,w nlsddfeecsi h uniiso tyr- of quantities the in differences analysed we study, this In ora fCl cec 2 (24) 125 Science Cell of Journal p75–GFP ( B n MDCK and ) SI ( C el eeqatfe,adbn neste eenraie oGAPDH. to normalized were intensities band and quantified, were cells ) p75 n MDCK and uui a enrpre o myobl detyr- for of reported accumulation been rapid microtubules has Comparatively detyr-tubulin-enriched tubulin curled. these frequently neuronal 2009), on al., are observations et with (Bisig agreement In cells 2011). al., et (Quinones incre microtubules authors detyrosinated The interactions. 1998). motor dynein (Johnson, tubule enhance the tubules may of B surface modifications pre-existing post-translational the these that of on speculate detyr- wall of facilitated outer the stretches in the Long found is been 1985). have Barra, which tubulin and described Detyr- detyr-tubulin, (Arce 1987). from microtubules been turnover al., to individual an et positive by has tyr- Webster generated isoforms. be double 1986; can microtubules two stretches al., of tubulin et the of length (Geuens of previously the labelling sections In of 1992). alternating Segmental isoforms most al., with detyrosinated et along and microtubules Brown tyrosinated present 1990; the were Black, both cells, and MDCK (Baas ends minus ssoni hssuyadb unnse l,tenme of number the al., et Quinones by and study this in shown As SI cells. ( A yae fMDCK of Lysates ) m m. ssa h el eoepolarized become cells the as ases Chlamydomonas p75–GFP ssclue ndifferentiation in cultured asts rMDCK or n 5 .( 3. lglu along flagellum SI D DKcells MDCK ) el transfected cells tubulin. Journal of Cell Science fti eraeo pcltafcigi MDCK in in trafficking reduction apical the on 20–30% secretion participation decrease that the this gp80 observed exclude of cannot unlikely or we the However, is transport tubulin. from assembled p75 it exclusively apical Moreover, results in plausible. reduction seem dramatic not does ee fasmldtblnwti h eetdrneaffect range detected the the MDCK in polarized within variations which in tubulin Do 2006), trafficking process. assembled al., membrane this et of microtubule of (Peris cell- level complexity of CLIP-170 addition, absence the as or In indicates such presence the 2011). factors by specific modulated al., be of can et rates be dynamics growth (Szyk average cannot the ratios microtubules cells, reduced seeding osteosarcoma These overexpression human dimers. in TTL after situation where tubulin the with of days accordance in sequestration are 3 the and by measured assembled explained between tubulin ratios converse soluble However, enzyme. the o eeteeae eeso y-uui nMDCK did we in as tyr-tubulin simple, of not in is levels story TTL elevated the that detect of al., seems not detyr-tubulin It amount et in 2004). decrease al., the Mialhe a et Increasing being (Kato 2004; in results 2006). al., of accordingly expression cells et al., feature HEK293T (Kato et common protein cells Soucek a cancer 2001; TTL is of the types detyr-tubulin low several a increased Indeed, of by of towards microtubules. paralleled balance detyrosinated scenario, assembly the in the opposite the in pushes decrease shift elevates and a relative that microtubules tyr-tubulin argue could Barra, tyrosinated towards and One Arce 1980). 1978; al., equilibrium al., et et Deanin (Arce 1985; preferentially microtubules reaction on detyrosination place the takes and preferentially tubulin, acts soluble TTL on microtubules. detyrosinated of depletion eoclswsfudt eacmaidb nices in increase an by accompanied be (Bre to found detyrosination the was or data, microtubule MDCK between cells our interactions cell with Vero for to cell agreement mechanism extensive of In development detyr-tubulin-dependent transformations. a morphological suggests al., which et (Chang detyrosination 2002), microtubule of differentiation inhibition morphological by This blocked was 1984). al., et (Gundersen medium Sy ta. 01.Ti cnrosest rvi npolarized in prevail to the seems scenario with microtubules This complex into MDCK 2011). elongated incorporation al., its an et prevent tubulin forms (Szyk may dimeric and it to dimer 1978) efficiently tubulin binds al., TTL Since et that cells. (Arce shown MDCK in been tubulin has polymerized it to soluble of ratio day, the 1 of study. interval this time in detected rise a initial seeding the within by reflected cells is which individual or subconfluent omsnhmltasto ET nimraie human immortalized in (EMT) epithelial- transition during detyrosination tubulin and to-mesenchymal in morphology increase cellular An on function. effects tyr- dramatic have assembled modifications of pool 1984). large al., considerably et (Gundersen the tyr-tubulin tubulin soluble by of amount covered the being in are alterations are that it equilibrium be Alternatively, well the might mechanisms. cellular in additional by changes compensated that suggesting possibly n ic h eieyo w aoaea ag rtiswas proteins cargo basolateral two of MDCK delivery in 1998), unaffected the al., et since (Grindstaff to cells proteins and MDCK of of transport membranes the plasma in both involved be to appear microtubules nte ur shwTLoeepeso a euti the in result can overexpression TTL how is query Another neetnl,ordt ugs htTLoeepeso affects overexpression TTL that suggest data our Interestingly, eetees aitosi h qiiru ftetotubulin two the of equilibrium the in variations Nevertheless, TTL–GFP el,wihmiti osatyhg eesof levels high constantly maintain which cells, TTL–GFP el,agnrlefc ntrafficking on effect general a cells, ´ ndtrtblnu o2dy after days 2 to up detyr-tubulin in ta. 91 nconfluent, in 1991) al., et TTL–GFP TTL–GFP TTL–GFP el?Since cells? cells. cells, pteilitgiyaddtrtblndpein6005 depletion detyr-tubulin and integrity Epithelial hne uigclua ifrnito navreyo cell of epithelial variety into MDCK tubulin-tyrosination differentiation monolayers. a the of perturbs overexpression in induction TTL by artificial differentiation Reciprocally, cellular level types. morphological the for during required in is increase degree changes sudden specific a a to conclusion, detyr-tubulin In of 2006). interphase al., during et polarization (Peris cell cells and spindle mitosis both endothelial in during defects positioning in in to results detyrosination fibroblasts tubulin TTL-null reattachment excess mammalian However, 2010). enhanced al., alterations, et in (Whipple morphological results promotes which cells epithelial mammary ako oiiycudb xlie yadpeino detyr- of depletion MDCK a in by explained microtubules be stable could tubulin-enriched mobility The morphology. of polarized prematurely lack a acquire and islands cell nrae lgtyi hstm nevl 0%ices nTTL in increase 100% even A and interval. time high this the constantly in whereas slightly remains 5, increases day tyr-tubulin to 2 of day concentration from decreases detyr-tubulin of level in islands partners thus cell neighbouring form is with vicinity. down, contacts It their settle it cell-to-cell 1988). cells as develop Bulinski, cell immobile and and a these of that (Gundersen asymmetry likely, migrate developing to the for begins needed are which iei oosd o oepeeetal ln dynamic motors and molecular along guide function that microtubules the in preferentially modified on translationally cilia data sensory move Recent apical of 2009). stability not al., the that et (Cai to do considering microtubules especially transport motors cells, Golgi MDCK kinesin of as post domain serve kinesin-driven detyr-tubulin,membrane microtubules for intracellular detyrosinated stable in tracks that alterations raft- suggests detyrosinated present by soluble, and which motor modulated this of be expression along can In trafficking This proteins TTL 2008). cargo apical non-associated traffics al., that 2010). or et associated demonstrate and (Dunn we Jacob, cells report trans-Golgi live to and in also following microtubules binds is (Astanina events Kif5c preferentially trafficking motor exit kinesin apical network the in that involved shown We 2007). al., previously et (Jaulin from surface have proteins cell membrane the to of network delivery trans-Golgi isoform the apical anti-kinesin kinesin-1 for are essential the in is Particularly motors KIF5b export. observed kinesin endosome that in was suggests involved the compartment which cells, collapse recycling in antibody-injected A 2002). al., the role et (Lin of surface a facilitate cell and the to play compartments transport vesicular recycling microtubules endocytic of detyrosinated these organization In cytoskeletal cells. stable CHO as from cells, came microtubules trafficking intracellular detyrosinated for tracks epithelial for polarized Evidence fully cells. in microtubules detyr-tubulin-positive (Erck cells, 2005). mice anomalies neuronal al., in morphogenetic of et display role polarization neurons vital the TTL-null a for since plays required TTL essentially basic of is a and at expression the detyr-tubulin addition, epithelial in of In in concentration level. TTL decrease the of maintain a amounts layers al., constant cell by et that (Whipple indicates detyr-tubulin characterized in This increase 2010). is an and cells TTL this and induction of synthesis cancer EMT for effect Similarly, reason breast the detyr-tubulin. a of reverses in as levels TTL high regarded in of be results knockdown can period since same observation, the in levels nplrzdtredmninlmnlyr fMC el,the cells, MDCK of monolayers three-dimensional polarized In h usinaie bu h ucino eann olof pool remaining a of function the about arises question The TTL–GFP .elegans C. el r esmbl,asml into assemble mobile, less are cells upr h dao post- of idea the support TTL–GFP cells, Journal of Cell Science DKclswr utrda 37 at cultured immunoprecipitation were surface cells and MDCK transfection siRNA culture, Cell Methods and Materials the integrity. in epithelial modifications of tubulin maintenance of and function post- establishment an the provide of of should on picture interactions pattern studies integrated their Further and the cells. involved MDCK in components formation in all changes trafficking monolayer membrane that affect and of, of time, modifications show balance tubulin first because We translational the the metastasis. tubulin interest cell for change promote in wider here, EMT thus of and alterations in tyrosination is of tubulin occur cells that role epithelial pathomechanisms the in of modification understanding of greater morphogenesis 2008). the al., for et (Spiliotis required cells MDCK is polarized pol transport and of vesicle coupling post-Golgi (Liao septin-2 characterized that these be filaments GTP-bind shown small further cross-bridge with can alignment intermediate the addition, tracks by Cytoskeletal in specific 1998). can, Gundersen, intracellular with it in because involved microtubules merely events not to transport is Kinesin-binding 2009). Yet microtubules Fischer, 2006). and detyrosinated for (Zekert al., required extension is et hyphal and fast (Reed microtubules detyrosinated from neurites to UncA, of binds preferentially kinesin-3 subset motor, specific kinesin transport a and another to binding kinesin-1 cargo promotes of acetylation which in cells, trans intracellular in to microtubules we guidance cells. Therefore, intracellular these of in 2010). domains assist membrane Kreitzer, specific factors the additional and that as (Jaulin propose were tubulin predominantly acetylated shown A for microtubules poles. detected previously cell be two tyrosinated could the accumulation of subapical or one in to detyrosinated focused involved not specifically did we that apart motors microtubules, However, of observe kinesin concentration 2010). subapical al., general of a et alternative from the regulation (Hammond an trafficking to is the intracellular Autoinhibition contributes for 2009). Setou, even instrument mammalian and in which (Konishi polarity thus neuronal cells 2010), of would maintenance and Kneussel, code’ motors establishment the of ‘tubulin and activity regulate or localization This (Janke the modifications regulate motors. that tubulin signposts kinesin-2 provide The these of 2011). that al., velocity et propose (O’Hagan destinations authors subcellular specific to 6006 hr,clswr iue to diluted were cells short, hme.TredmninlMC elcsswr bandb sn BD using Neubauer by a obtained in were counted cysts were Cells cell seeded. MDCK Matrigel were Three-dimensional filter chamber. six-well per quantities ern ta. 08.Iae eeae napopomgrwr nlsdwith analysed were phosphoimager (Cramm- (Bio-Rad). a software on before ONE generated QUANTITY described Images the 2008). as al., essentially et Behrens performed were immunoprecipitation loecne o h eeaino oaie ooaeso DKor MDCK of the monolayers TTL–GFP polarized expressing of and 90% stably least generation antibiotics at line for the with neomycin cell MDCK containing For supplemented medium a in fluorescence. FCS selected of were cells 10% generation transfected containing For medium Eagle’s glutamine. modified glucose) Dulbecco’s in or g/l serum (4.5 calf foetal 5% containing (PAA) 2m oesisligi elcluedse,cvrdwt eimatrgelling after 37 medium at with days covered 7–10 dishes, culture for cell incubated in and lying 30 coverslips environment. mm cold 12 a in Matrigel of lsi n ml nefrn N sRA rnfcino DKclswas cells MDCK manufacturer’s MDCK the of of to labelling transfection according Biosynthetic (Invitrogen) (siRNA) instructions. 2000 RNA Lipofectamine with interfering performed small and Plasmid fnwysnhszdmtra ntetasGlintoka 20 at network trans-Golgi the in material synthesized newly of repcieo h udmna nih neihla ilg,a biology, epithelial in insight fundamental the of Irrespective of modifications post-translational of role the for evidence Further TTL–GFP TM aeetMmrn arxPeo-e-re(DBocecs.In Biosciences). (BD Phenol-Red-Free Matrix Membrane Basement ora fCl cec 2 (24) 125 Science Cell of Journal cells , 3 6 , 10 2 6 7 10 ˚ el e 0c utr iho equivalent or dish culture cm 10 per cells ne %CO 5% under C 4 ˚ el/lPSadmxdwt h aevolume same the with mixed and PBS cells/ml ,i %CO 5% in C, m otcmsfo utrdneuronal cultured from comes port fti itr eesra u ncold on out spread were mixture this of l guayae irtblsto microtubules yglutamylated n etn.Siitse l have al. et Spiliotis septins. ing p75 rMDCK or 2 2 nmnmmesnilmedium essential minimum in ihrglrmdu change. medium regular with seglu nidulans Aspergillus SI el,accumulation cells, ˚ n surface and C , i5 Acm n niZ1(ye) h oolnlatbde anti-E- antibodies monoclonal Biosciences The BD from purchased (Zymed). were (6C5) anti-ZO1 anti-GAPDH and anti-CD29 and cadherin, (Abcam) Kif5B TT-3 GCAAAGUCAUU-3 e cecsd uiy asil,Fac)adG jka SaeUiest of University USA). (State York, (Faculte Ojakian New anti-SI, generously Bivic G. Center, Le and Science were monoclonal A. France) Health Switzerland), York Marseille, antibodies The Basel, New Luminy, (Biozentrum de anti-gp135 (GAPDH). Hauri Sciences P. des and Ltd H. by (ME20.4) HyTest provided anti-p75 and monoclonal CD29) (E-cadherin, oylnlatbde eeue:at-T PoenehGop,anti-Claudin-1 following Group), The (Proteintech imaging. GSDIM anti-TTL anti-clusterin- and used: immunoblots (Invitrogen), were for polyglutamylated antibodies used the was polyclonal recognizing tubulin not of AdipoGen) (Millipore) form antibody (GT335, tubulin detyrosinated tubulin polyglutamylated detyrosinated anti-detyrosinated Cruz), Santa polyclonal (YL1/2, tubulin anti- anti-acetylated monoclonal and used: 1A) were antibodies tubulin following The Antibodies 5 (1) – duplexes two following ACCUUGGAAAGUAGUUU-3 TTL the of depletion with specific performed experiments, (RNAi) was interference RNA-mediated For interference RNA cDNA TTL of RevertAid generation (Fermentas by specifications followed (Qiagen) manufacturer’s Kit the Mini MDCK to from RNeasy isolated according was the RNA total using the plasmid cells TTL-containing a of construction For constructs DNA efre n4 aaomleyefr1–5mntsfloe ya by followed was fixation minutes the 10–15 analysis for TTL For paraformaldehyde hour. 1 blocked 4% and for minutes in 5 powder/PBS++ for methanol milk performed ice-cold with 1% fixed were in E-cadherin cells Claudin-1, the modifications, ZO-1, tubulin and of analysis immunofluorescence For staining Immunofluorescence 4 after at 8.0) incubated pH Tris- X-100, mM times, Triton (25 0.5% buffer indicated desoxycholate, lysis sodium in the 0.5% collected NaCl, were mM cells 50 the HCl, lysates cell of procedures preparation analysis For protein and assays Biochemical 0mntsa 37 at minutes 10 r-amdPS HM2Mgyeo/%Tio -0 5 MPPS 0mM 50 PIPES, MgCl mM mM (50 2 EDTA, X-100 mM Triton 1 glycerol/2% HEPES, M PHEM/2 PBS, pre-warmed tp ihPSpu MCaCl washing several mM 1 After plus proteins. presentations. PBS surface ratio with biotinylate for S) steps to and Scientific) P (Thermo of polymerized Biotin (sum of tubulin Values total Tubulin blots. by software. identical divided 1D from The were LabImage (P) GAPDH pellet. the tubulin to tubulin using normalized calculated polymerized were was the signals ratio from dimer separated to was polymer fraction tubulin soluble ivrsand h ape eenraie opoietlst bands. lysate prominent to normalized were and samples SDS-PAGE time The by by indicated separated stained. tested were the the silver was lysates after content replacing cell gp80 quantification, then by The For cells, medium. immunoblotting. analysed this a relevant concentrating was the and using of gp80 removing intervals medium stained of basolateral secretion and and The apical SDS/ SDS-PAGE procedure. with biotinylated by eluted staining were the silver were separated proteins select lysates the buffer, cell steps to washing the dithiothreitol-containing Scientific) renewed BSA (Thermo After with proteins. beads Together surface X-100. neutravidin Triton with 1% incubated EDTA, mM 5 NaCl, EE,p ..Teclswr ahdoc npewre eim resuspended 37 medium, at pre-warmed in recover once to washed left were and cells The 6.1. pH HEPES, ta. 03.Clswr avse nnplr,3o as(oa)atrseeding, after (37 (polar) days medium 5 or pre-warmed 3 in polar), (non resuspended 1 and harvested were centrifuged Cells software. 2003). imager 1D al., LabImage gel et using Intas quantified an secondary were and results Scientific) horseradish-peroxidase-conjugated The Fischer camera. (Thermo with CCD ECL by performed visualized antibody was Detection B+ ln,teclswr rznat frozen were cells the alone, PBS++ ircluoemmrns h ebae eebokdi %simdmilk 4 skimmed at 5% overnight incubated in and to hours blocked 1 transferred for were PBS and in membranes powder Biotech) The Pharmacia membranes. (Amersham nitrocellulose system Hoefer-Mini-VE the 12,000 at minutes 5 h DAwsue stmlt naPR(owr T rmr 5 primer: TTL (forward PCR a in template the using as vector pEGFP-N1 into used cloned was gaattcatgtacaccttcgtggtg-3 (5 cDNA primer TTL reverse The a using transcriptase) reverse sacnrl uieaesRAwsue (5 used was siRNA luciferase control, a As o ufc itnlto el eeicbtdwt h rslne Sulfo-NHS- crosslinker the with incubated were cells biotinylation surface For irtbl sebywssuidesnilya rvosypbihd(Ligon published previously as essentially studied was assembly Microtubule 9 /5 9 -UCGAAGUAUUCCGCGUAC+GTT-3 a tblnatbd Snpi ytm) h oylnlanti- polyclonal The Systems). (Synaptic antibody -tubulin ˚ .Floigacnrfgto tp( iue,8000 minutes, (2 step centrifugation a Following C. g 9 /3 n h uentnswr eaae ySSPG using SDS-PAGE by separated were supernatants the and a 9 9 ˚ -UUACAAACCUAACGUUUCAGU-5 tbln(im-lrc) oolnlanti-tyrosinated monoclonal (Sigma-Aldrich), -tubulin o 5mnts fe w diinlwsigsesin steps washing additional two After minutes. 15 for C n ees T rmr n h C rdc a then was product PCR the and primer) TTL reverse and a g8;SnaCu) anti- Cruz), Santa (gp80; 9 /3 a 2 tbln(ilpr)admncoa anti- monoclonal and (Millipore) -tubulin 9 -UUGUAAUGGAACCUUUCAUCA-5 n MMgCl mM 1 and 2 H69 a de,mxdadicbtdfor incubated and mixed added, was 6.9) pH , ˚ narttn ltom etiue for centrifuged platform, rotating a on C Eco 2 20 Iand RI ˚ n ye n2 MTi,10mM 150 Tris, mM 20 in lysed and C 9 -CGUACGCGGAAUACUUCGA- 9 Bam ). 9 -ggatcccccagcttgatgaacgcagc-3 2 Irsrcinsites. restriction HI PS+/. lcnand glycin M (PBS++)/0.1 ˚ ihseii antibodies. specific with C b ctnn(im) anti- (Sigma), -catenin ˚ )cnann 0mM 30 containing C) a 9 tbln(ln DM (Clone -tubulin -UGUUUGGAUU- 9 2 5 (2) , TM 9 M-MuLV -CAUU- 9 g . the ) a 9 9 ). ´ - - Journal of Cell Science rw,A,Sagtr .adBak .M. M. Black, and T. Slaughter, A., Brown, Bre R. Caputto, and A. C. Arce, A., J. Rodriguez, S., H. Barra, nioy2 or,scnayatbd 4hus ocs 34 0minutes, 30 primary 33342 hours, Hoechst 2 hours, blocking 14 each. minutes antibody minutes, 20 20 secondary steps the longer washing fixation with hours, with but 24 manner methanol Hoechst blocking labelled antibody similar with a stained times: in antibody in were Nuclei stained secondary incubation performed hour. were FCS/ 1 cysts with was for Three-dimensional 5% PBS++ 33342. and antibody in in dye blocking hours primary AlexaFluor 2 hour indicated 1 with for and incubation reagents/PBS++ minutes) (5 The acetone PBS++. in step permeabilization ii,C . hsa .E,Zma,G . ur,S . atne,V .adArce, and S. V. Santander, A., S. Purro, G., G. Zampar, E., M. Chesta, G., C. Bisig, M. M. Black, and W. P. Baas, R. Jacob, and K. R. Astanina, Caputto, and S. H. Barra, A., J. Rodriguez, E., M. Hallak, A., C. Arce, S. H. Barra, and A. C. Arce, E. K. Mostov, and A. L. Katz, G., Apodaca, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.109470/-/DC1 online available material Supplementary R.J.]. to Graduiertenkolleg 593 R.J., Sonderforschungsbereich and to R.J., 1033 to JA Forschungsgemeinschaft, 1216 numbers Deutsche [grant the Germany by Bonn, supported was work This Millicell the Funding with careful help for for for and Lamp Przyborski technical B. advice (Millipore). J.M. and for Voltohmmeter for manuscript ERS-2 protocol, Ligon Ackermann the L.A. ratio W. of and reading tubulin and Holz the Dienst P. providing M. thank to We assistance. grateful are We Acknowledgements MDCK resistance, transepithelial the of determination the For measurement TER 20 a using B DMI6000 Leica a a using on microscope recorded AOBS SP2 were TCS experiments Leica a on 40 acquired were images Confocal Microscopy MDCK experiments Time-lapse otaei obnto ihteVlct mgn otaepackage software imaging Volocity the with Leica combination the with (Improvision). performed in to was according evaluation (Leica) software Data system super-resolution specifications. GSD B manufacturer’s SR 100 DMI6000 the Leica Leica APO a was a using PL using acquired (TIRFM) HCX were 2010) an microscopy al., et with fluorescence (Schneider microscope before reflection described internal as performed total The objective. sdfrmcocpctm-as experiments. time-lapse microscopic for 100 used with medium of replacement Dish or fe edn,temdu a hne n E esrmn was measurement TER in and (Millipore) Voltohmmeter changed ERS-2 was Millicell the medium using duplicate. hours the 24 every seeding, performed after hours 8 MDCK 6 867-882. cells. epithelial and fibroblastic in detyrosination tubulin and ocnrtdi h rxmladdsa ein fgoigaxons. growing of regions distal and proximal the in concentrated formation. neurite preclude not does cells CAD in ribonuclease-insensitive a A. C. by a C)tyrosine by C)arginine (14 (14 proteins and own system. its system into incorporates that ribonuclease-sensitive brain rat from preparation stability. and composition both in differ that domains hnllnn n h feto h noprto fteeaioaiso tubulin on cells. acids MDCK of amino and trafficking these tyrosine of release incorporation to the and of incorporate effect assembly. to the microtubules and and phenylalanine tubulin of Capability state. steady at endosomes. microtubules recycling apical via is cells MDCK in IgA ,M . epro,R,Kes .E n asni E. Karsenti, and E. T. Kreis, R., Pepperkok, H., M. ´, H i lnpcrmtojcie(ec irsses.Telv-eltime-lapse live-cell The Microsystems). (Leica objective planapochromat oil iloWls n nuae until incubated and Wells) Willco , TTL–GFP GFP 20) ako tblzdmcouue sarsl fteasneo ao maps major of absence the of result a as microtubules stabilized of Lack (2009). .Neurochem. J. rMDCK or .Neurochem. J. el eesee nofles nuae t37 at incubated filters, onto seeded were cells TTL–GFP 20 31 97-108. , el o.Lf Sci. Life Mol. Cell. 205-210. , 18) ees fCtria yoiefo uui and tubulin from tyrosine C-terminal of Release (1985). el eesee ngasbtoe ihs(WillCo dishes glass-bottomed on seeded were cells ice.J. Biochem. 21) I5,aknsnmtrivle napical in involved motor kinesin a KIF5C, (2010). 19) niiulmcouue nteao oss of consist axon the in microtubules Individual (1990). m EE-otiigmdu h el were cells the medium HEPES-containing M 6 226 19) el sebe irtblsare microtubules assembled Newly (1992). , 19) eetrmdae rnctssof transcytosis Receptor-mediated (1994). betv.Tehg eouinimages resolution high The objective. 05%cnlec 2hus.After hours). (2 confluency 40–50% 311-317. , 67 1331-1342. , ESJ. FEBS .Cl Biol. Cell J. .Cl Biol. Cell J. 19) ellrinteractions Cellular (1991). 276 il Cell Biol. 7110-7123. , 17) soluble A (1973). ˚ .Cl Biol. Cell J. 111 ,adstarting and C, 6 125 495-509. , 71 r fluotar dry 67-86. , 149-160. , GFP (1978). 119 and , pteilitgiyaddtrtblndpein6007 depletion detyr-tubulin and integrity Epithelial ei,L,Tey . Faure M., Thery, L., Peris, rk . ei,L,Adiu,A,Misrl . rbr .D,Vre,M., Vernet, D., A. Gruber, C., Meissirel, A., Andrieux, L., Peris, C., Erck, Molina-Parı B., and T. Liverpool, A. E., Bivic, E. Le Morrison, S., H., Dunn, Leffler, E., Salomonsson, A., Koch, W. C., M. Greb, Gordon, D., and Delacour, K. R. Hanson, F., S. Preston, R. G., Jacob, G. and Deanin, M. Dienst, I., C. Cramm-Behrens, io .adGnesn .G. G. Gundersen, and G. Liao, ’aa,R,Paek,B . iv,M,Pik,P,Nue,K . al .H., D. Hall, C., K. Nguyen, P., Phirke, M., Silva, P., B. Piasecki, R., O’Hagan, Mu Lafaneche A., Mialhe, R. F. Maxfield, and G. G. Gundersen, X., S. L. Lin, E. Holzbaur, and M. Tokito, S., S. Shelly, A., L. Ligon, hn,W,Wbtr .R,Slm .A,Gue,D,Paa,A,Esrc,J .and P. J. Eiserich, A., Prasad, D., Gruber, J. A., A. K. Salam, Verhey, R., D. and Webster, W., E. Chang, Meyhofer, R., J. Martens, P., D. McEwen, D., Cai, oih,Y n eo,M. Setou, and Y. Konishi, rnsaf .K,Bcla,R .adNlo,W J. W. Nelson, and and L. C. R. Bacallao, J. K., Bulinski, K. Grindstaff, F., Cornelissen, R., Nuydens, G., G. Gundersen, G., Geuens, hwj,S,Gnesn .G n uisi .C. J. Bulinski, and G. G. Gundersen, S., Khawaja, Imai, T., Ozaki, Y., Nakamura, M., Ohira, A., Nakagawa, K., Miyazaki, C., Kato, A. G. K. Johnson, Kreitzer, and E. Rodriguez-Boulan, X., Xue, F., Jaulin, udre,G . ansi .H n uisi .C. J. Bulinski, and H. M. Kalnoski, G., G. Gundersen, C. J. Bulinski, and G. G. Gundersen, aln .adKete,G. Kreitzer, and F. Jaulin, J. K. Verhey, and D. Cai, W., J. Hammond, ak,C n nusl M. Kneussel, and C. Janke, Y. H. J. Naim, and K. M. Verhey, Alfalah, M., and Heine, D. R., Jacob, Cai, V., Soppina, L., T. Blasius, W., J. 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