n hrb ly rca oei h raiainadfnto fthe of function and organization organization the Golgi. in role crucial Golgi a results plays in thereby These and participates trafficking. MMG8 ER-to-Golgi that in indicate was the functioned On and molecules. EB1 MAPRE1), two whereas these as of known localization Golgi, Golgi (also the EB1 for required tracking end h ag hoZagadRbr .Qi* Z. Robert and Zhang Chao Wang, Zhe transport Golgi ER–Golgi in and functions organization isoform microtubule myomegalin identified newly A ARTICLE RESEARCH ß 4904 2014 September 1 Accepted 2014; April 17 Received ([email protected]) correspondence for *Author Kowloon, Bay, Water Clear Technology, China. and The Kong, Science Neuroscience, Hong Molecular of of University Laboratory Kong Key State Hong and Science by Life of followed Division is it and sites, ER-exit cargo at are of vesicles packaging COPII-coated the by into Golgi, initiated ER- is The cells, the proteins carriers. of post-Golgi transport to by into to-Golgi sorted (ER) eventually used reticulum are they endoplasmic pathway where the secretory al., from et the transported (Miller cells featuring In of structure center ribbon 2009). the continuous near a positioned During ministacks form Golgi . to of Golgi the stages and enable Golgi late the from the derived during reassembly, reassembles then ribbon-like fragmentation undergoes and the Golgi crescent-moon-shaped surrounds the divide, typically cells that a When . region perinuclear exhibits the in Golgi morphology the cells, and Rios Su intact 2003; an 1998; Bornens, require (Lippincott-Schwartz, Golgi cytoskeleton the of microtubule in function role and The pivotal positioning transport. a assembly, and plays sorting that modification, organelle post-translational membranous protein a is Golgi The INTRODUCTION trafficking Protein Myomegalin, Microtubule, Golgi, WORDS: KEY MMG8 Furthermore, fragmentation. with Golgi (ER)-to-Golgi associated caused reticulum and endoplasmic Disrupting proteins. with trafficking affected both interaction of expression this stability and the MMG8 for AKAP9), required as was known AKAP450 (also 8 AKAP450 interacting variant with by networks myomegalin cis-Golgi to isoform, predominantly localized identified (MMG8), newly protein This muscle characterized a used myomegalin. poorly identify the the We of to isoform obscure. spectrometry expressed underlying widely mass remain with Golgi mechanisms coupled the immunoprecipitation the of and organization organization However, microtubule its microtubule- trafficking. for major microtubules a protein be requires to that known site is organizing cells mammalian of Golgi The ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,40–97doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. c tblncmlxsmdae irtbl nucleation, microtubule mediated complexes - c tblncmlxsadwt h irtbl plus- microtubule the with and complexes -tubulin telnadClni 00.I nepaeanimal interphase In 2010). Colanzi, and ¨tterlin ulaina h og osntrqiecnrsms n it and centrosomes, require not does on Golgi cellular instead depends all the of at pigment microtubule Moreover, retinal 2007). half nucleation al., human et almost (Efimov in cells Golgi example, RPE1 epithelial the For from 2009). originate 2009; microtubules al., al., et Miller et 2007; al., Rivero et Efimov 2005). 2001; al., al., et et (Chabin-Brion Watson 1997; with al., Golgi associate et Scales the that 1997; towards al., microtubules carriers et (Presley along cargo move ER–Golgi to dynein–dynactin of formation the irtbl ulao ncls hc xssi h omof form the in exists which cells, in nucleator microtubule sAA9 KP5,C-A n yein n GMAP210 and hyperion) and CG-NAP AKAP350, AKAP9, as ( complexes as nw sTI1)hv enpooe ob novdin involved be to proposed been have TRIP11) as known (also 00 oge l,20;Fn ta. 09.Hr,w eotta a in that functions report MMG8, we Here, isoform, 2009). MMG al., nonmuscle al., et identified et Fong newly 2008; (Choi al., tips et microtubule Fong 2010; growing in at and regulation centrosomes on a microtubule protein organization microtubule is microcephaly-related In in that involved human muscles. is present that a skeletal protein CDK5RAP2, and only of the heart homolog is in MMG1 expressed databases, is GenBank 4D-interacting that phosphodiesterase protein a kDa The as 2001). a al., to et (Verde as referred protein was cloned thus was 4D and phosphodiesterase NP_055459) nucleotide cyclic with accession interacted that protein GenBank proteins MMG1; EB unknown. 1, other remains and Golgi microtubules the EB1 at Although whether function targets and Golgi, 2008). localize subcellular the with Steinmetz, to associate attachment and microtubule (Akhmanova and microtubule regulating plus- including dynamics microtubule functions, the diverse at +TIPs EB1 perform the with ends EB and association tips, the in microtubule localized of EB1 are growing member that all of prototypic at serine-rich detected the domain is these EB1, and (EBH) family, 2009). of homology al., interaction basic et the end-binding (Honnappa of for by the required class with surrounded is one +TIPs that motif present In is 2009). SxIP sequences al., an weaker et other considerably +TIPs, (Komarova the exhibits from them, activity distinct to tip-tracking be relative and, to MAPRE3, proteins appears two and MAPRE2) as MAPRE1 EB2 known as whereas (also properties, known tip-tracking among (also similar proteins, display EB3 respectively) (EB) and end-binding EB1 the all which with almost interact plus-ends, must microtubule track +TIPs To 2008). (Akhmanova Steinmetz, (+TIPs) proteins and tracking plus-end called proteins 2009). of al., et Rivero 2009; al., et and (Miller trafficking motility directional assembly, cell ribbon Golgi for Golgi required the are from originating Microtubules 2012). Golgi-associated al., the et Vinogradova in (Rı thus microtubules and of Golgi nucleation the to recruitment tubulin h og evsa ao irtbl-raiigcenter microtubule-organizing major a as serves Golgi The nayattohbi cen ymgln(ymglnisoform (myomegalin myomegalin screen, two-hybrid yeast a In group diverse a accumulate microtubules of tips growing The c us.TecsGlipoen KP5 as known (also AKAP450 proteins cis-Golgi The TuCs). c tbln(fmve l,20) h principal the 2007), al., et (Efimov -tubulin ´ se l,20;Rvr ta. 2009; al., et Rivero 2004; al., et os mmg1 euneecdsa encodes sequence c -tubulin , 230- c -

Journal of Cell Science 1) ugsigteeitneo mle sfrso proteolytic in or isoforms detected smaller of were In existence the and bands suggesting S1D). S1D), protein fibroblasts the Fig. lower to cells, addition two material epithelial myotubes, (supplementary C2C12 proliferating cells of neuroblastoma extracts of unless results; using identical obtained were yielded below 532C. shown results antibodies the otherwise, 532C antibody, specified The second MMG8. sequence and of terminus A carboxy 443M on the S1C). SxIP against based generated Fig. EB1-binding was 532C, material an predicted and (supplementary were domains motif coiled-coil that include MMG8 analysis of features raiain hc srqie o fiin ER-to-Golgi microtubule efficient Golgi for in required role key is enable a which to trafficking. plays EB1 ER-to-Golgi organization, MMG8 tethering efficient in and Therefore, also capture that and microtubule-tip AKAP450. recruiting protein nucleation with in interacting involved expressed by microtubule is Golgi widely MMG8 the Golgi, a of the side On is cis the MMG8 to targets that Our reveal trafficking. ER-to-Golgi results and organization microtubule Golgi ARTICLE RESEARCH n 2,rsetvl.MG oooswr lofudin found also were homologs MMG8 gallus 98% Gallus respectively. of similarities 92%, sequence and overall exhibited that NP_835181) and in homologs region and detected the NP_001126198) product, were contains RT-PCR MMG8 that the acids of MMG8 to amino isoforms. corresponding 1116 MMG of 637–925 the protein number among possesses a MMG8 terminus encodes 27, accession carboxy human exon the unique of exons the of splicing a alternative 27 of the exon under consists of spliced Because MMG8 alternatively GenBank the of and sequence in 11–26 coding sequence The protein the HQ333476. deposited this have we of MMG8; as designated 13 isoform, hCG1755149. and to NP_001002811 peptide unique by sequence shared the Specifically, residues to 9 hCG1755149 last 1C). cDNA the (Fig. the from by extended encoded hCG1755149 sequences matched and sequence peptides NP_001002811), four accession sequences last (GenBank the the 5 matched variant sequences peptide MMG material spectrometry the from (supplementary of sequences Most mass peptide S1B). 13 Fig. Tandem To of total mass- 1B). 1A). a perform (Fig. to (Fig. protein revealed gels from the analysis 2001) band immunoprecipitated the al., spectrometric excised we and et cells band, HeLa (Verde from protein MMG1 this of identify The size rabbits. of expected band immunizing single a for detected protein , 443M, was used as recombinant designated was product the antibody, resulting and bacteria RT-PCR bacteria, from the in purified sequence, expression the this for and generate cloned against To S1A), sequencing. antibody Fig. through an verified material was (supplementary product amplified cells extracted RNA HeLa total MMG the from This from databases. amplified specifically in was found sequence variants MMG large other in RT-PCR human cultures, the targeting mmg1 cell primers oligonucleotide proliferating using in performed was proteins MMG detect To MMG8 of Identification RESULTS organization. Golgi and trafficking 5 D nHL xrcs hsi usatal mle hnthe than smaller substantially is This extracts. HeLa in kDa 150 M8wsdtce sasnl rti adi immunoblots in band protein single a as detected was MMG8 ae ntepoensqecn aa ecoe oe MMG novel a cloned we data, sequencing protein the on Based euneecdn mn cd 7–6,argo present region a 474–762, acids amino encoding sequence , eou tropicalis Xenopus , 5-D pce splmnaymtra Fig. material (supplementary species 150-kDa u musculus Mus og abelii Pongo and ai rerio Danio Gnakaccession (GenBank Gnakaccession (GenBank c ust promote to TuCs h structural The . mmg . uigctknss M8apae nbt og twins Golgi Golgi. both the of in side 1D), appeared (Fig. cis TGOLN2) the 1D). MMG8 (Fig. on as cytokinesis, resides known trans- During MMG8 the (also of that staining TGN46 the suggesting known Golgi with 1D). (also protein merge (Fig. the GM130 not protein did Golgi on cytoplasm it cis-Golgi signal but the GOLGA2), the of MMG8 as that the to weak in similar of a was we detected and distribution Golgi, the was MMG8, the Moreover, in staining antibody. of enriched MMG8-specific highly general localization extracts. was the tissue staining with subcellular MMG8 heart cells was rat HeLa the in it immunostained determine antibody although 443M S1D), To the cell Fig. by these of material recognized any in (supplementary detected not cultures was MMG1 However, products. eandi h R(i.2) y4 i fe hfigt 32 to shifting after min 40 By 2D). (Fig. ER the in retained atr fVV.Tepoenepesda 40 at expressed protein of The delivery VSVG. the of pattern in MMG8 of Golgi. role the a to indicates resistance VSVG endo-H which of 2C), acquisition (Fig. the delayed markedly depletion og n ag muto h rti en ipre nthe in dispersed being protein the of the at amount present large being in a amount distribution and small mixed Golgi a a with cells displayed cells, control VSVG in MMG8-depleted contrast, Golgi By the 2D). at (Fig. localized predominantly was VSVG akrudi hc og atrswr o eetdwhen detected not were patterns Golgi which ( in RNA background protein with used target transfected the we to designed depleted MMG8, were small of Two oligonucleotides mmg8 expression. (si)RNA MMG8 function suppress interfering to the (RNAi) interference investigate To ER-to- and trafficking organization Golgi Golgi in functions MMG8 rfikn,w sdtewl-hrceie ag VSVG cargo 40 well-characterized At glycoprotein). the ts045 virus used stomatitis (vesicular we trafficking, of integrity structural the maintaining a Golgi. in indicate the MMG8 they for thus MMG8 role and when altering material crucial organization, that even Golgi show (supplementary results perturbs these levels cytotoxicity expression together, Taken high and S2). Fig. moderately fragmentation low at whereas Golgi at expressed distribution, levels of caused expressed pattern low Golgi transiently it a at exhibited when MMG8 MMG8 MMG8 structures; of levels, overexpression of Golgi the that expression determined affected also ectopic We be 2B). could the (Fig. fragmentation Golgi by This overlapped 2B). 2B), rescued that (Fig. (Fig. patches nuclei into with broken antibody these largely were In ribbons anti-MMG8 staining. Golgi anti-MMG8 the the of cells, specificity with the confirmed labeled which were cells srvril ifle n hsacmltsi h R however, 32 ER; the to in cells accumulates shifting thus and misfolded reversibly is eitn fe 0mna 32 at endo- min became 40 VSVG after of amount (endo- resistant oligosaccharides al., substantial H H a endoglycosidase et Golgi, cells, by control Scales cleaved medial In be H). 1997; the cannot VSVG al., to to et linked delivered along (Presley transport When subsequent Golgi and 1997). the ER the to from microtubules export its in results M8epeso i o fetteepeso n ER and expression the affect 40 at not Disrupting VSVG 2C). of did (Fig. accumulation min expression 60 after MMG8 resistant became VSVG total ocrooaeteefnig,w xmndtedistribution the examined we findings, these corroborate To et ots hte M8fntosi ER-to-Golgi in functions MMG8 whether test to Next, n h rnfcino ihrsRAit el effectively cells into siRNA either of transfection the and , ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal mmg8 ˚ lostepoent odcrety which correctly, fold to protein the allows C treigsRA xiie weak a exhibited siRNAs -targeting , 5 euto,Fg A.Cells 2A). Fig. reduction, 85% ˚ ˚ Fg CD.Hwvr MMG8 However, 2C,D). (Fig. C ,adapoiaeyhl fthe of half approximately and C, ˚ a effectively was C ˚ ,VSVG C, 4905 ˚ C,

Journal of Cell Science EERHARTICLE RESEARCH 4906 in MMG8 and AKAP450 Reciprocal coimmunoprecipitated the of AKAP450. quantification with tested specific Schmidt coimmunoprecipitated we AKAP450 MMG8 were and 2009; 1999), MMG8 that al., of al., showed et experiments et association immunoprecipitation Witczak 1999; Rivero potential al., 1993; et that through the large-scale Takahashi al., protein identified 1999; cis-Golgi et al., were In et a (Keryer subunit EB1 is PKA MMG8. and AKAP450 regulatory anchors (PKA) Because to the spectrometry. A mass bind kinase both protein MMG8, that sought of we of proteins function, immunoprecipitates MMG8 identify into insights to mechanistic gain AKAP450 To with MMG8 of Association microtubule-dependent a in the 2E,F). occurs in (Fig. manner. involved that expression is trafficking MMG8 MMG8 that ER-to-Golgi indicate of results using silencing these performed Collectively, the was was by VSVG assay of unaffected trafficking transport ER-to-Golgi the the cells, When (Cole nocodazole-treated sites 1996). ER-exit al., at localize et which stacks, 2D). functional into (Fig. Golgi networks the ER-like fragments and depolymerization puncta microtubule Nocodazole-induced of form the in cytoplasm lyadpootoal Fg 3A); (Fig. proportionally and ally pcfcdsrbto atr Fg E.Teerslsso htthe that any show display results These not 3E). did by (Fig. pattern 3E); fragment distribution (Fig. 389–1116 specific Golgi expressed the the at the contrast, specifically levels, low localized at 1–389 expressed Golgi fragment When or MMG8. of binding- on examined brefeldin-A and fragments also AKAP450-binding deficient the AKAP450 We with of distribution S3). subcellular with cells Fig. the material treating colocalized (supplementary the fragments by after MMG8 double- their Even dispersed 3D). nocodazole, performed verified (Fig. was and Golgi we the Golgi AKAP450 Next, at and colocalization AKAP450-binding 3C). prominent MMG8 (Fig. the of of MMG8 impaired deletion immunostaining the of strongly moreover, 1–389 3C); activity AKAP450, (Fig. acids to AKAP450 to amino binding 389–1116 bind acids for amino not comprising comprising did sufficient fragment MMG8 MMG8 was of the region whereas 1–389 head acids The amino tested. with was coimmunoprecipitation AKAP450 their stoichiometric and expressed a ectopically were 0.98 formed was extracts. AKAP450 MMG8 cell the to and in AKAP450 complex of MMG8 showed ratio 3B) Therefore, molar (Fig. dye the fluorescent that a with stained gels SDS-PAGE ompteAA40bnigst,vrosMG fragments MMG8 various site, AKAP450-binding the map To ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal nagdo h ih.Saebars: Scale 5 right. the on are enlarged areas Boxed and proteins. MMG8 Golgi for stained of cells cells) HeLa 100 of (100% micrographs Representative (D) spectrometry. mass using analyzed P1–P13 peptides color. denote same the in shown are regions sequence Identical variants. MMG the of representation A schematic (C) spectrometry. mass through of , protein The Blue. with Coomassie stained or anti-MMG8 with immunoblotted and SDS-PAGE using resolved were immunoprecipitates The (IP). for immunoprecipitation used were buffer RIPA in extracts prepared HeLa (B) (PS). preimmune serum the or (443M) anti-MMG8 antibody an with probed gels) were 15% and (6% SDS-PAGE using cis-Golgi novel a protein. is MMG8 1. Fig. 5 D a xie o analysis for excised was kDa 150 m m. A eaetat resolved extracts HeLa (A) 6 0.1:1.

Journal of Cell Science EERHARTICLE RESEARCH ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal tvrostmsdrn nuainat incubation 32 during times various at extracted were cells with The transfected VSVG–YFP. were cells HeLa (C) ersnaieof are representative that The micrographs right. shows the figure on enlarged are regions boxed The (FLAG–MMG8). siRNA-resistant construct an of by transfection rescued the was MMG8 of knockdown The microscopy. using immunofluorescence analyzed were siRNA targeting or control with transfected ftepoenby protein the of expression the suppressed si- MMG8-2) and (si-MMG8-1 siRNAs of MMG8 transfection The siRNAs. targeting or control with cells transfected HeLa of Immunoblotting (A) transport. ER-to-Golgi efficient Golgi and for organization required structural is MMG8 2. Fig. 5mna 32 at for min incubation 15 after fixed were that ( cells of images eemnd n uniaiedt from data quantitative and were determined, VSVG the of to amounts relative total VSVG of form endo-H- the resistant of amounts The VSVG. check for to immunoblotted were the samples assay, the Following resistance. t40 at 10 with treated were VSVG–YFP expressing Cells (E,F) shown. phenotypes condition the each exhibited for analyzed the cells of 200 90% least of At min immunostained. 40 or 0 at 32 fixed were Cells mean (D) the as here presented are experiments independent three h mean the show data experiments; three independent (from times collected various cells at of (E) endo-H- assay the resistance of results the figure shows The further. incubated and ˚ ˚ n eeasydfrendo-H for assayed were and C nuainadthen and incubation C ˚ o n hnsitdt 32 to shifted then and h 6 for C 6 .. n representative and s.d.) ˚ F.Saebr:5 bars: Scale (F). C , . , 5.()RE cells RPE1 (B) 85%. 0 f10cells) 100 of 90% 00cls( cells 1000 m /lnocodazole g/ml mmg8 mmg8 6 s.d. n 5 - - 4907 m 3). m. ˚ C

Journal of Cell Science EERHARTICLE RESEARCH og oaiain eue Nit elt h rtisoea a at 4908 one proteins the deplete to RNAi used we Golgi- localization, Golgi and AKAP450-binding the MMG8. of as domain targeting serves sequence 1–389 ots hte M8adAA40rqieec te for other each require AKAP450 and MMG8 whether test To otree rti seFg ) M8dpeinddnot did depletion MMG8 4). the Fig. the of with (see degradation treated the protein were prevent to AKAP450 nontargeted MG132 or protein. inhibitor MMG8 other proteasome the of of localization depleted Golgi Cells the checked then and time ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal ape cl as 5 bars: each Scale for sample. analyzed cells 100 of muolte o h M8proteins MMG8 the for immunoblotted were inputs and immunoprecipitates The immunoprecipitation. anti-FLAG to subjected were FLAG–MMG8 encoding constructs expressing cells HEK293T (C) to 0.98 AKAP450 was of MMG8 ratio molar the that showed experiments independent three from Data quantified. were AKAP450 of and intensities MMG8 Ruby Sypro The immunoblots (IBs). on detected Sypro or with Ruby stained were MMG8 with coimmunoprecipitated Proteins (B) experiments). independent (three hw nEFwr bevdin observed were E,F in shown were phenotypes cells The The immunostained. h. then 12 for MG132 treated with were RNAi using was depleted AKAP450 or in MMG8 cells which HeLa (F) right. the on are enlarged areas boxed The Golgi marker. a and proteins the transfected for examined The were cells. cells HeLa in (GFP) expressed ectopically were fragments right. MMG8 the (E) on enlarged is boxed area The MMG8. and AKAP450 for double-stained were cells RPE1 (D) AKAP450. and (anti-FLAG) nus aaso h mean the show respective Data the inputs. of that to proteins relative precipitated the of the amount shows graph The samples. the quantifying and immunoblotting by analyzed were inputs and proteins The immunoprecipitated extracts. HeLa using performed were (IPs) immunoprecipitations anti-AKAP450 and Anti-MMG8 (A) AKAP450. Golgi with the interacting to by targets MMG8 3. Fig. 6 0.1:1. m m. 6 s.d. . 90%

Journal of Cell Science eue muoltigt rb h eeso ohproteins. both of levels the probe to immunoblotting used we MMG8-independent that through is and Golgi AKAP450 MMG8 the to mechanisms. binding that to its localizes indicate through AKAP450 Golgi results the Golgi these to the whereas targeted Collectively, to AKAP450, localization 3F). of MMG8 (Fig. localization inhibited depletion Golgi AKAP450 the affect markedly ARTICLE RESEARCH fMG nmcouuenceto.Imnpeiiainof Immunoprecipitation nucleation. microtubule in involvement potential MMG8 the the explored at we of 2009), nucleation al., microtubule et (Rivero for Golgi required is nucleation AKAP450 microtubule Because Golgi-based in becomes functions protein MMG8 other the proteasome. proteins, the the by degraded of is one and unstable of Thus, absence 4B). the (Fig. treatment in MG132 following In were altered AKAP450 4B). markedly and (Fig. not MMG8 cells of control levels treatment in the the cells, level MG132 control-transfected of the of level approached timecourse the eventually the AKAP450, and over or increased MMG8 protein either the other of depleted on cells pathway. depends proteasome-dependent In a protein through the whether degraded test each were to proteins next applied of was MG132 other. expression the proportional of expression the a 4A), that (Fig. versa to vice revealing and AKAP450 led of level protein expression the in reduction MMG8 suppressing Notably, nteRA xeiet efre nMG n AKAP450, and MMG8 on performed experiments RNAi the In M8coprecipitated MMG8 8 euneo M8i nipnal o t idn to binding its for indispensable is MMG8 of sequence 389 can MMG8 that with conclude associate any we Therefore, show 5C). not (Fig. did activity binding 389–1116, fragment, MMG8 MMG8 extracts, control the both towards activity In binding of similar cells. extracts showed with AKAP450-depleted performed assay and pull-down control a in used was in MMG8 immunoprecipitated and AKAP450 and from c dissociated was cells MMG8 which in in buffer, RIPA expressed reciprocal ectopically was the to bind In can of GCP3, MMG8 component 5A). with core (Fig. coprecipitated specifically another examined was MMG8 we experiment, that components bevduiga nioyta eonzsmlil ptpsof epitopes multiple recognizes that c antibody an using the observed endogenous date, To expression. of MMG8 silence staining to RNAi used we MMG8, n lob nacn h tiigsga yuigtandem using by signal staining the enhancing fixation before by cytosol the also extracting be by and cannot problem it Therefore, this that staining. tackled levels cytosolic we low background such above at detected Golgi readily the at present is tubulin eodr niois nclsetatdwt saponin-containing a with extracted cells In antibodies. secondary us(upeetr aeilFg 4.Teimmunoprecipitated The S4). Fig. material (supplementary TuCs tbln(Rı -tubulin oeaiewehrteGlilclzto of localization Golgi the whether examine To ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal ´ se l,20) ecniee h osblt that possibility the considered We 2004). al., et os c usidpnetyo KP5 n htte1– the that and AKAP450 of independently TuCs c usi h bec fAA40 MMG8 AKAP450, of absence the in TuCs c tblnadGP,wihare which GCP2, and -tubulin c tblna h og a nybeen only has Golgi the at -tubulin he needn experiments. independent three n KP5 r lte sthe as mean plotted are AKAP450 MMG8 and of amounts The immunoblotted. and prepared cell were which extracts after MG132, were with cells treated siRNAs, with transfection mean after the as shown experiments are independent three from extracts. Data control the in amounts the respective of percentages as expressed were si-MMG8- cells of si-AKAP450-transfected extracts and the in AKAP450 MMG8 of and amounts the quantification, After immunoblotted. were siRNAs with indicated transfected the cells RPE1 of Extracts (A) stability. for are dependent AKAP450 mutually and MMG8 4. Fig. c us(i.5) ots whether test To 5B). (Fig. TuCs 6 ..uigtedt uniidfrom quantified data the using s.d. c us(i.5) whereas 5C), (Fig. TuCs 6 c ..()A 2h 72 At (B) s.d. usrequires TuCs c u core TuC c c TuCs. TuC- 4909 c -

Journal of Cell Science EERHARTICLE RESEARCH ooaie ihMG tteGli(i.5) M8ddnot did MMG8 and 4910 5D). centrosomes, (Fig. Golgi the the at at MMG8 detected with Golgi colocalized being the that to showed addition GM130 intact. and largely MMG8 was for labeling buffer, c tblnwsraiyosre tteGliin Golgi the at observed readily was -tubulin c -tubulin fcls n Nimdae elto fMG eliminated of MMG8 staining centrosomal of of the depletion attachment RNAi-mediated Golgi the population and large cells, a in of localization centrosomal prominent display ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal ihsRA eeimnsandfor immunostained were siRNAs were with cells RPE1 (D) for pull- immunoblots. double-stained The on that siRNA. examined cells AKAP450 were of or downs lysates control RPE1 with with transfected together were assay used pull-down then a were in beads The buffer. RIPA were in cells immunoprecipitated HEK293T in expressed (389– transiently FLAG–MMG8 1116) and were FLAG–MMG8 inputs (C) and immunoblotted. immunoprecipitates using The performed extracts. were HeLa (IPs) immunoprecipitations (B) GCP3 with c associates MMG8 5. Fig. uniiaino h sltdGlimmrnsa the as membranes protein Golgi mean the isolated shows the were graph of (WCLs) The quantification lysates immunoblots. cell the whole on the analyzed and Both fractions cells. Golgi siRNA-transfected the from 5 isolated bars: were Scale membranes sample. ( each representative for are analyzed D,E centrosomes. in indicate shown Arrows Images II). (Man II mannosidase u tahett h Golgi. the to attachment TuC 6 ..fo he needn xeiet;** experiments; independent three from s.d. c tblnwtotdsenbyaffecting discernibly without -tubulin c tblnadMG.()Clstransfected Cells (E) MMG8. and -tubulin c tbln(i.5) oevr the Moreover, 5E). (Fig. -tubulin AB niMG A n anti- and (A) Anti-MMG8 (A,B) c usadi eurdfor required is and TuCs c tblnand -tubulin m . .()Golgi (F) m. 0)o 0 cells 200 of 90%) P , 0.01.

Journal of Cell Science eahetof detachment ARTICLE RESEARCH og.T etti osblt,w xrse h EB1-binding-deficient the expressed we the to possibility, attachment this for test MMG8 To with Golgi. interaction its on trafficking depend might ER-to-Golgi EB1 in functions EB1 Golgi-associated Similar which 7F). 7G). nocodazole, (Fig. (Fig. with fragmentation treated manner Golgi cells at induced MMG8-dependent using localized obtained an clearly were preserve EB1 results in but cells, Golgi Golgi buffer extracted proteins a the the the cytosolic with In visualize remove networks. extracted Golgi to were To cells saponin 7E). clearly, (Fig. tip- containing the EB1 EB1 affect of markedly of Golgi- localization not did the behavior it but eliminated tracking EB1, expression of pattern MMG8 associated speckles Inhibiting tip-tracking microtubule these 7E). as (Fig. detected Notably, being AKAP450. to to addition 7C,D). MMG8 in (Fig. of binding EB3 the affect to and not contrast did EB1 substitutions striking towards In binding no alanines. exhibited activity Leu311 mutant to motif, L311A/P312A MMG8 the SxIP changed MMG8, the an wild-type were of constructed residues Pro312, crucial The We and two 7B). which (Fig. in PKA. the mutant MMG8 in of to this conserved and counterparts region, is sequence addition by 298–329 a its motif verified contains within MMG8 in motif was 7A). immunoprecipitates SxIP (Fig. putative MMG8 immunoblotting EB1 anti-EB1 with the of means EB1 contained that of coimmunoprecipitation MMG8 revealed of spectrometry EB3 and EB1 Mass with MMG8 of Association orltdwt h eoa fGliascae MMG8 the Golgi-associated for required of of attachment is of centrosomal MMG8 localization that removal indicate Golgi results These the 5F). (Fig. with correlated P1clsatrcl-nue irtbl depolymerization. in microtubule performed cold-induced were after assays cells regrowth RPE1 , ute upr h ocuinta M8i eurdfor required data is These MMG8 Golgi. 6C). the (Fig. that at region organization conclusion and Golgi growth the the microtubule the microtubules support from the reduced and further radiate 6C) (Fig. substantially not Golgi did the the 6C). expression at and (Fig. density microtubules pattern, microtubule MMG8 of radial density high a Suppressing a in harbored region distributed Golgi were and centrosomes in ministacks cells; 6B). the (Fig. from control cells grow the MMG8-depleted the to the in failed from MMG8 microtubules for the grew however, co-stained cytoplasm. were microtubules the that in washout, ministacks dispersed nocodazole were ministacks Following Similar Golgi which nucleation. that cells, centrosomal RPE1 showed affect nocodazole-treated using depletion not obtained does the were MMG8 results but rewarming; Golgi by nucleation of the microtubule inhibits affected at min depletion MMG8 2.5 not Thus, 6A). after (Fig. was even regrowth Golgi of centrosomal the microtubule min from exhibit 6A). 2.5 not (Fig. growth did centrosomes At cells MMG8-depleted the to the from 6A). contrast, also (Fig. observed By and Golgi min were the 1 from filaments emanate for microtubule rewarmed prominent rewarming, were after aster cells centrosomal a as the and Golgi control the at a appeared either microtubules with transfected an were cells or the assays, the Before nRE el,E1wsimnsandtruhu h Golgi, the throughout immunostained was EB1 cells, RPE1 In nRE el,mcouue mntdfo h og and Golgi the from emanated microtubules cells, RPE1 In microtubule in functions MMG8 whether investigate To mmg8 treigsRA ncl-rae oto el,short cells, control cold-treated In siRNA. -targeting c tblnfo h og ebae was membranes Golgi the from -tubulin c usbti ipnal o the for dispensable is but TuCs c TuCs. og abelii Pongo and u musculus Mus efre h SGtafcigasy sn el htwere to that shifting after cells min 15 using At 32 constructs. assays MMG8 we the trafficking EB1, with Golgi-associated transfected VSVG of specifically function the Therefore, EB1 the detached test 8A). performed To (Fig. mutant Golgi. Golgi MMG8 mutant the the from the the of of expression expression contrast, the and the by By AKAP450 affected wild-type 8A). of the (Fig. in localization localization cells EB1 the MMG8-transfected with mutant the compared with as transfected substantially cells protein, the was in EB1 eliminated of and even localization or Golgi diminished Golgi, The 8A). tip-tracking mutant microtubule the (Fig. EB1 the the of affect at and markedly not localization wild-type did transfections prominent the Both morphology showed Golgi analysis. protein in for changes moderate obvious selected wild-type at exhibit proteins were not the the did expressed and and that cells levels (L311A/P312A) The cells. MMG8 in protein of version mutant M8de o oti h conserved the contain However, EB3. MMG8 not and does fibroblasts EB1 CDK5RAP2, MMG8 with and interacts that Like cells motif SxIP epithelial S1D). the contains in Fig. detected (Verde material not muscles is (supplementary was in that 2001), expressed protein al., is a et and MMG1, CDK5RAP2 spliced. the to multiply by homologous is encoded which isoform detected gene, cultures only MMG fibroblast the in and was conserved MMG8 epithelial is examined, proliferating protein the this that In MMG8 suggesting vertebrates. Golgi. mammals, in addition the in those zebrafish to on and frog chicken, organization in its identified microtubule demonstrated were homologs and in MMG8, role identified protein, we crucial study, novel this Golgi a In characterized 2012). the al., and an et of Vinogradova into 2009; positioning al., stacks et subcellular cisternal (Rı microtubules the Golgi-associated of on depend and assembly Golgi the integrated cells, mammalian In DISCUSSION eut niaeta h eoa fE1fo h og mar the impairs VSVG. Golgi These the of 8C). from transport EB1 (Fig. ER-to-Golgi of MMG8 removal the wild-type that indicate with results transfected mutant cells EB1-binding-deficient in the than with transfected more cells resistance in endo-H mutantslowly acquired EB1-binding-deficient VSVG Consequently, the 8B). expressing (Fig. the in cells dispersed of still was cytoplasm VSVG but MMG8, wild-type expressing 01 aaah ta. 02,weesMG a neatwith interact can MMG8 whereas 2002), with al., associate et Takahashi to 2011; known is networks. functional function cis-Golgi AKAP450 at such a nucleation One mutually microtubule form functions. mediating involves was various likely performing stability proteins when complex their these the and Therefore, in dependent. complexes present mostly subcellular nocodazole-induced were same AKAP450 after and S3). MMG8 ministacks Fig. brefeldin-A Remarkably, material after Golgi (supplementary MMG8 depolymerization sites at microtubule view, ER-exit and this at treatment AKAP450 Supporting with AKAP450. colocalized with interaction ER-to-Golgi its efficient and organization microtubule trafficking. in the at functions Golgi MMG8 functions, and organization involved centrosomal is is in CDK5RAP2 which with Whereas region, CDK5RAP2. amino-terminal interacts to its homologous not MMG8 through Golgi instead, the to 2010); localizes al., 2008; al., et et Wang (Fong and CDK5RAP2 amino of the respectively, terminus, at carboxy present are that domain centrosome-targeting the M8i i-og rti htlclzsa h og through Golgi the at localizes that protein cis-Golgi a is MMG8 ˚ ,temjrt fVV a oaie oteGlii cells in Golgi the to localized was VSVG of majority the C, ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal c tblnwsntmarkedly not was -tubulin c u-idn oanand domain TuC-binding c us(utd tal., et (Hurtado TuCs ´ se l,20;Rivero 2004; al., et os c usand TuCs 4911

Journal of Cell Science EERHARTICLE RESEARCH 09 niaeta h M8AA40cmlxanchors 4912 complex MMG8–AKAP450 al., et the Rivero 2011; that c al., et indicate (Hurtado others 2009) of those and study this c 37 to cells the Golgi. warming the by at initiated nucleation was microtubule regrowth for microtubule required depolymerization, is MMG8 6. Fig. h loecneitniiso irtblswr esrda h og raadi h yols.Atrsbrcigtebcgon,terto of ratios the background, the subtracting After cytoplasm. the in t on and enlarged area mean are Golgi areas the represe boxed the show The shown at Data siRNAs. images measured with derived. the transfected were were cells A,B, microtubules Golgi:cytoplasm of In of micrographs depolymerization. Immunofluorescence intensities (C) nocodazole-induced fluorescence condition. after The each performed for analyzed was cells regrowth 100–200 of Microtubule (B) microtubules. MTs, tubulin). ust i-og ewrs hr hyncet microtubules. nucleate they where networks, cis-Golgi to TuCs together, Taken 5C). (Fig. AKAP450 of independently TuCs 6 ..( s.d. n 5 0clsfrec uniiain.Cr,cnrl cl as 5 bars: Scale control. Ctrl, quantification). each for cells 30 A P1clswr rnfce ihteidctdsRA.Atrcold-induced After siRNAs. indicated the with transfected were cells RPE1 (A) ˚ .Teclswr hnsandfrMG,TN6admcouue (anti- microtubules and TGN46 MMG8, for stained then were cells The C. eosre eetv irto fMG-eltdclsi a thus, cell in cells and, finding, MMG8-depleted directional this of AKAP450 with migration agreement impaired defective in eliminating observed 2009); al., we of is et (Rivero effects migration microtubules the Golgi-derived of One ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal m m. nt eright. he . 90% a -

Journal of Cell Science EERHARTICLE RESEARCH i.7. Fig. e etpg o legend. for page next See ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal 4913

Journal of Cell Science agtn h a at-LGI) h muorcpttsadipt were inputs and immunoprecipitates the by IP), proteins (anti-FLAG the tag (311/ immunoprecipitating the mutant After targeting L311A/P312A expressed. its ectopically and were MMG8 2A) black (C) in NP_835181. highlighted MMG8, (asterisks). residues mouse binding EB1; EB1 with residue) for interaction acid crucial for amino are any required denotes is (x that SxIP indicated motif the the contains with MMG8 immunoblotted Golgi. (B) were the antibodies. the inputs to and and recruitment performed (IPs) its was immunoprecipitates mediates MMG8 and of EB1 Immunoprecipitation to (A) binds MMG8 7. Fig. ARTICLE RESEARCH 4914 bound their with to together directly CDK5RAP2, AKAP450 Golgi, and the c on protein MMG8 scaffold a recruits as functioning by to that binds AKAP450 whether c However, assay. scratch-wounding 5 bars: all Scale for condition. shown each images for The right. represent the experiments The on immunofluorescence immunostained. enlarged were are to cells areas Cells nocodazole the boxed (G) with which right. treated after the were microtubules, on siRNAs depolymerize enlarged indicated are the buffer areas with saponin-containing boxed transfected a The with fixation. extracted before either were (F) were or cells (E) The indicated TGN46. untreated and the left EB1 with MMG8, transfected for triple-stained cells were (anti-GFP) HeLa siRNAs EB3 (E,F) for (anti-FLAG). (IB) MMG8 The immunoblotted and constructs. were MMG8 inputs the and and GFP–EB3 immunoprecipitates HEK293T with (D) transfected MMG8. doubly were wild-type cells WT, vector; FLAG Vector, immunoblotted. t seta oei h og tahetof attachment Golgi the in the role of essential attachment its Golgi the promote could ipnal o h etooa oaiainof localization centrosomal the for dispensable Therefore, nuigtesrcua nert fteGli is,MG is MMG8 First, Golgi. the recruiting of for integrity required structural the by ensuring activities Golgi-nucleated Golgi-associated trafficking various microtubules. of Golgi-associated in in regulating that MMG8 suggest disruption MMG8 participates observations of these MMG8 to role Collectively, The the excluded. direct response be trafficking. cannot a of in ER-to-Golgi although microtubules, decreased in because the role was thus silencing the crucial efficiency and to a envision microtubules transport EB3 We of 8). plays or (Fig. attachment Golgi Golgi EB1 of EB1/EB3-mediated the with the to function EB3 that or binding this EB1 of its Interestingly, association required 2C,E). (Fig. results MMG8 Our 2005). trafficking ER-to- al., microtubule-dependent et for Golgi Watson required (Presley is 1997; MMG8 al., dynein–dynactin that association et showed Scales motor in 1997; Golgi al., minus-end-directed et the the towards move with cargos secretory which of attachment targets. the subcellular mediate to +TIPs EB1 tips that other microtubule is notion SxIP-motif- with the with formed an with function agree results complexes STIM1, our a together (Grigoriev Therefore, to 2008). membranes al., ER Such EB1 EB3 et with associates of membranes. that and binding protein containing Golgi the EB1 of to tethers reminiscent observations MMG8 tips These MMG8 8A). of microtubule (Fig. that mutant Golgi the suggest EB1-binding-deficient centrosomal from the EB1 that the observation delocalized their the of by underlying supported expression is This that manner. dependent from distinct localization. is that us nte cnroi htbt KP5 n M8bind MMG8 and AKAP450 both that is scenario Another TuCs. is scenario potential One unclear. remains MMG8 through TuCs M8i novdi eea ciiista otiueto contribute that activities several in involved is MMG8 on microtubules requires trafficking ER-to-Golgi Efficient MMG8- an in Golgi the to localized EB1 that determined We c c us uhitrcin of interactions such TuCs; usaettee oteGlitruhamechanism a through Golgi the to tethered are TuCs c ust,adtu nbigmicrotubule enabling thus and to, TuCs m m. og abelii Pongo . c uswti h complex the within TuCs 0 of 90% c M8 NP_001126198; MMG8, us ncnrs to contrast In TuCs. c us M8was MMG8 TuCs, , c 0 el analyzed cells 500 us(i.5E). (Fig. TuCs eu n eemitie t37 bovine at fetal maintained 10% were containing medium and in serum cultured were cells Mammalian culture Cell (His hexahistidine a with fusion in 926–1116, expression and bacterial 637–925 for acids cloned amino were containing fragments, MMG8 Two Antibodies 2009). AACUUUG- al., (si-AKAP450, et AKAP450 (Rivero human AAGUUAACUAUCAA) was against duplex synthesized siRNA also an si- AAGCAGAGAGACAGCUCUAUA); AACCUCCAGUGGCUGAAAGAA; MMG8-2, (si-MMG8-1, synthesized PCR- number were MMG8 of duplexes target accession means siRNA to by under Two MMG8 mutagenesis. GenBank into site-directed in based introduced were deposited Mutations been MMG8 HQ333476. have carboxy- sequences corresponding nucleotide MMG8 a and the protein of replacing The with RT-PCR. by using NP_001002811 amplified constructed sequence of was The sequence Japan). MMG8 Chiba, terminal of (Kisarazu, Institute obtained sequence Research was coding 5) DNA variant Kazusa (MMG the NP_001002811 from clone MMG human The oligonucleotides and Plasmids METHODS AND MATERIALS a.Tercmiatpoen eeprfe sn Ni using purified were proteins recombinant The tag. als X,at-ansds I(ilpr,Bleia A n anti- Biotechnology, and UK). MA) Oxford, Cruz Kidlington, Billerica, Serotec, Santa (Millipore, polyclonal, (sheep (FL, II TGN46 anti-mannosidase anti-GFP TX), anti- were Aldrich), Dallas, purchased following antibodies (Sigma polyclonal The The FLAG MO). 2008). Louis, (St anti-GM130, al., Aldrich NJ); Lakes, sources: anti- et (Franklin Biosciences commercial and BD Fong from from anti-EB1 2010; and were anti-AKAP450 The described al., antibodies purified. been AKAP450 et has monoclonal was antibodies the (Choi and anti-GCP3 against and previously 1924–2170 the anti-GCP2 generated acids of using was generation amino Using by antibody containing membranes. sera an fragment nitrocellulose the methods, on from similar immobilized purified antigens 532C, were and respective against 443M as Antibodies generated designated Antisera were respectively. 926–1116 rabbits. and 637–925 immunizing acids were for amino and the used urea dialysis, M were After 6 (PBS). proteins saline of phosphate-buffered presence against the dialyzed in then CA) Valencia, (Qiagen, resins acid B.TeeMG ucin r eurdfrefcetER-to- efficient for required are functions MMG8 These EB3. AKAP450, by with Golgi complexes the functional on forming organization microtubule regulates MMG8 a which caused Golgi Golgi-localized MMG8 of the homeostasis MMG8. of the S2). maintaining organization of require Fig. to structural appears material proper overexpression (supplementary ensuring Golgi 1999). Therefore, the the al., structural of We et and disorganization 2010). that al., biogenesis Witczak et Mavillard Golgi 1999; observed 2006; for al., al., (Schmidt et required (Bejarano et reorganization PKA is which Takahashi as PKA 2007). AKAP450, 1999; Notably, such al., stabilizing et and al., molecules Marra for et regulatory proteins 2008; required several al., of is recruits et maintenance (Diao MMG8 delivery and structure Fourth, assembly Golgi the the ER-to-Golgi the for perturbs of required affects are MMG8 thus that al., membranes of et and Vinogradova knockdown 2009; transport the al., Third, et Golgi fragments (Miller 2012). Golgi and of positioning gathering assembly central for Golgi and for the clusters the facilitating larger into for at ministacks During required originating Golgi. are the microtubules centrosome to tips biogenesis, and microtubule EB1 Golgi attaches with thereby interacts and MMG8 Second, EB3 Golgi. the on, nucleation og rfikn n tutrlognzto fteGolgi. the of organization structural and trafficking Golgi olciey h aapeetdhri upr oe in model a support herein presented data the Collectively, ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal a tbln anti- -tubulin, c tblnadat-LG(2 rmSigma from (M2) anti-FLAG and -tubulin ˚ ne %CO 5% under C 2 c ea HEK293T, HeLa, . usadE1or EB1 and TuCs 2+ –nitrilotriacetic- 6 )

Journal of Cell Science hntps BC SGYPwscepesdwt h M8cntut oeaut rti rfikn.Terpeettv mgssoni r fcel of are B in shown images representative The trafficking. protein evaluate to constructs MMG8 the with coexpressed ( was VSVG–YFP (B,C) phenotypes. EERHARTICLE RESEARCH DE) ua eia imn pteilclshETRE (RPE1) hTERT-RPE1 cells epithelial medium pigment Eagle’s mouse retinal modified (a Dulbecco’s human C2C12 in (DMEM); and grown were line) cells cell line) neuroblastoma myoblast human (a IMR-5 MCF-7, ( cell analyzed Every right. the on enlarged are regions boxed The trafficking. (L311A/P312A). ER-to-Golgi MMG8 efficient 311/2A, for MMG8; required wild-type is WT, EB1 Golgi-associated 8. Fig. eaaeeprmnsadi hw stemean the as shown is and experiments separate . 0 f10cls t0o 5mnatrtase o32 to transfer after min 15 or 0 at cells) 100 of 80% 6 s.d. ˚ ,a niae.Saebr:5 bars: Scale indicated. as C, ehoois.CC2molsswr ifrnitdit ytbsby myotubes into differentiated (Life were MEM Carlsbad, in myoblasts grown Technologies, C2C12 were (Life fibroblasts Technologies). fetal-lung (1:1) human MRC-5 F12 and DMEM:Ham’s CA); in grown were A P1clstasetdwt M8adismtn eeimmunostained. were mutant its and MMG8 with transfected cells RPE1 (A) m .()Ed- eitneo SGwsqatfe sn aafo three from data using quantified was VSVG of resistance Endo-H (C) m. ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal n 5 0)soe h presented the showed 100) 4915 ls

Journal of Cell Science h ed eewse ihRP ufradwt yi buffer immunoblotting. lysis of for proteins means with bound tested by beads, examined the and then sedimenting were After were buffer Tween-20. beads 0.1% RIPA containing The with Tween-20. to binding 0.1% washed with immunoprecipitation, were After supplemented buffer. beads anti-FLAG-coupled RIPA in using the Aldrich) immunoprecipitated Sigma (M2, was beads cells HEK293T nonredundant in a searching by spectra. mass revealed tandem was with database Foster identity sequence Biosystems/Sciex, Protein quadrupole/time-of-flight CA). 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A-conjugated aee otscnayatbde n hndne anti-goat-IgG donkey Technologies). then (Life dye and same the antibodies with labeled cells Alexa-Fluor- antibodies secondary – secondary signals, antibodies goat staining secondary two enhance labeled with To sequentially stained fixation. were methanol before saponin) rsHlp .,10m al MET,1 rtnX-100, Basel, Triton (Roche, 1% cocktail EDTA, protease-inhibitor mM mM a [20 1 buffer and NaCl, lysis dithiothreitol mM in 150 mM prepared 1 extracts 7.4, pH cell from Tris-HCl against tags) antibodies peptide (using their immunoprecipitated were proteins Transfected assays Brefeldin-A, pull-down and Sigma serum. Immunoprecipitation from horse purchased were 2% MG132 with Aldrich. and cycloheximide supplemented nocodazole, DMEM using ARTICLE RESEARCH H74 . urs,5m DAadteprotease-inhibitor the and EDTA 4916 900 at mM Tris-HCl were centrifuged 5 mM were Cells (10 sucrose, homogenates 1989). The buffer M al., cocktail). 0.5 from homogenization adapted et 7.4, ice-cold was pH (Malhotra membranes in elsewhere Golgi homogenized isolating described for methods used protocol The membranes Golgi of Isolation a with transfected 40 were at cells cultured then siRNAs, and with VSVG–YFP transfection encoding after plasmid h 72 At assay trafficking VSVG methanol cold with at either min fixed 5 were for cells immunostaining, perform To microscopy Immunofluorescence with incubated with 4 were at preclearing agitation extracts After the clarified. Technologies), with (Life then NaCl, supplemented Protein-A–agarose mM and SDS) 150 cocktail 0.1% 7.4, protease-inhibitor and pH deoxycholate the Tris-HCl sodium mM 1% (25 NP-40, extracts buffer 1% cell RIPA in MMG8, prepared endogenous were immunoprecipitate To Switzerland)]. SG(yuigat-F) oaayetesbellrdsrbto of distribution at methanol subcellular with the fixed analyze were cells To the anti-YFP). VSVG, using (by VSVG O H69 lcrl MMgCl mM 5 glycerol, M 2 acid- 6.9, 1,4-piperazinediethanesulfonic pH M KOH (0.1 To buffer Germany). saponin-containing Jena, Zeiss, an Carl using visualize a META, or acquired (LSM510 Japan) microscope were Tokyo, confocal Nikon, images TE2000, cell (Eclipse microscope staining, epifluorescence After temperature. room immunostained. oldfr1 i.Atrbiig h ape eemxdwt U/ 1 with mixed and were buffer samples the sample boiling, SDS-PAGE After in min. collected 10 for were Ipswich, boiled Biolabs, cells England Briefly, (New assayed MA). protocol was manufacturer’s resistance the Endo-H immunostained. according or resistance endo-H for ntaetetafcigasy,teclswr hne oamdu that medium a to changed were cells 100 the contained assays, trafficking the initiate el eeicbtda 32 at incubated were cells noHadte nuae t37 at incubated then and endo-H ntepl-onasyof assay pull-down the In c c tblna h og,clswr xrce o 0mni a in min 30 for extracted were cells Golgi, the at -tubulin usi xrcso P1clspeae sn h yi buffer lysis the using prepared cells RPE1 of extracts in TuCs ˚ 2 o iha M8seii nioybudt Protein- to bound antibody MMG8-specific an with h 4 for C 20 m /lccoeiieadwspeamdt 32 to prewarmed was and cycloheximide g/ml ˚ rwt %prfradhd nPSfr1 i at min 15 for PBS in paraformaldehyde 4% with or C ˚ o aiu ie n hnete analyzed either then and times various for C c us LGMG rninl expressed transiently FLAG–MMG8 TuCs, ˚ o eoeimnbotn for immunoblotting before h 4 for C 2 MET n 0.1% and EGTA mM 2 , 2 20 ˚ o i n then and min 5 for C ˚ g vrih.To overnight. C o 0mnat min 10 for ˚ ;the C; m l 4 oe .B,Sik,N,Mrta . og .adLpict-cwrz J. Lippincott-Schwartz, and J. Song, Z. A., R. Marotta, Qi, N., and Sciaky, C. B., Dai, N. K., Cole, S. Sze, A., P., Drechou, Liu, C., K., Y. Settegrana, Choi, F., Perez, J., Marceiller, K., Chabin-Brion, a aee ntpo .5Mscoe( l isle n1 MTris-HCl mM 10 in dissolved 90,000 ml; at centrifuged (1 and sucrose 7.4) M pH 1.25 of top on layered was mdzl H68 0m C,05m MgCl mM 0.5 KCl, mM mM 50 (50 buffer 6.8, cytoskeleton-stabilizing pH a imidazole using to by prewarmed fixation was before that briefly medium in a incubated then 37 to and PBS transferred ice-cold were with cells 10 with cold-treated them 37 treating regrowth, by cells microtubule or placing h initiate 1 by for depolymerized ice completely on were microtubules Cellular regrowth Microtubule eaao . arr,M,Vg,L,Hdlo .adVlso A. Velasco, and J. Hidalgo, L., Vega, M., Cabrera, E., Bejarano, O. M. Steinmetz, and A. Akhmanova, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.155408/-/DC1 online available material Supplementary material Supplementary number [grant Trust Charitable TUYF the and TUYF12SC05]. ITCPD/ Kong; number Hong [grant Commission of Technology 17-9] and Innovation the SEG_HKUST05] Kong; Grant Hong of Equipment of China [Area Special of and Committee AoE/M-06/08 Program Grants Scheme Research University Excellence Basic the Key 2013CB530900]; T13- National number Scheme the [grant [General Kong; Research Council Hong Theme-based Grants of and Research 607/12R] 662612 the and from 662511 grants Fund by Research supported was work This Funding the wrote R.Z.Q. and the Z.W. conducted data. C.Z. the and analyzed paper. R.Z.Q. Z.W. and experiments. C.Z. the Z.W., designed experiments. R.Z.Q. and C.Z. Z.W., contributions Author interests. competing no declare authors The interests Competing Hong Technology, and Science assistance. Tam of technical Jason University for and Kong China) discussions Hong Kong, valuable (The for Sun Wang MI) Xulun Yanzhuang Arbor, and thank Ann also Michigan, We of reagents. (University (Oslo for Ørstavik Norway) Sigurd Hospital, and Adam PA) University Singapore), Pittsburgh, Biology, University, Cell Mellon and (Carnegie Linstedt Molecular D. of (Institute Hong Wanjin thank We Acknowledgements 05m fec urs ouin rprdi 0m rsHlp .)and 7.4) pH 90,000 Tris-HCl mM at 10 centrifuged sucrose in prepared M then solution, 0.5 sucrose and each was of 1.0 1.1, ml layer, with (0.5 overlaid sequentially 1.25-M-sucrose membrane was This ml) sucrose. the (0.6 M fraction 1.2 band with to adjusted white and interface aspiration a through collected the as appeared above CA). which Brea, immediately fraction, Coulter, smooth-membrane (Beckman crude rotor swinging-bucket The TLS-55 a using by urs,wr olce,dltd13wt 0m rsHl(H74 and 7.4) (pH Tris-HCl mM M 10 M–1.0 (180,000 centrifugation with 0.5 by 1:3 of pelleted interface diluted the collected, at were enriched sucrose, were which membranes, Golgi DA %PG00ad01 aoi)(vtiae l,1996). al., et (Svitkina saponin) microscopy. 0.1% immunofluorescence using and analyzed were PEG4000 Microtubules 4% EDTA, muoltiganalysis. immunoblotting ˚ ormv uliaditc el.Tepsncerspraat( ml) (1 supernatant postnuclear The cells. intact and nuclei remove to C 19) og ipra uigmcouuedsuto:rgnrto fGolgi of regeneration sites. disruption: exit reticulum microtubule endoplasmic peripheral during at stacks dispersal Golgi (1996). complex. ring gamma-tubulin the by Biol. nucleation microtubule stimulates Pou organelle. and organizing G. Durand, tutrlsaiiyadboeei eedo soitdPAactivity. PKA associated on depend biogenesis Sci. and stability structural tips. microtubule of fate 309-322. the controls network protein ˚ ˚ .T euebcgon yolsi tiig el eeextracted were cells staining, cytoplasmic times background several reduce washed To C. were cells Nocodazole-treated bath. water C 119 191 ora fCl cec 21)17 9441 doi:10.1242/jcs.155408 4904–4917 127, (2014) Science Cell of Journal 3764-3775. , 1089-1095. , o.Bo.Cell Biol. Mol. s C. ¨s, g 20) h og ope samicrotubule- a is complex Golgi The (2001). o . t4 at h 2.5 for g 12 o 0mna 4 at min 90 for g 2047-2060. , o 0mna 4 at min 60 for 20) rcigteed:adynamic a ends: the Tracking (2008). m ˚ /lncdzl o .To h. 2 for nocodazole g/ml nteTS5 oo.The rotor. TLS-55 the in C 2 MET,01mM 0.1 EGTA, mM 1 , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. o.Bo.Cell Biol. Mol. ˚ na ultracentrifuge an in C ˚ )adte sdin used then and C) 21) CDK5RAP2 (2010). 20) Golgi (2006). 7 631-650. , .Cell J. .Cell J. 9 ,

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