nesetas 7 H88 rulne,Switzerland. Kreuzlingen, CH-8280 47, Unterseestrasse [email protected]) ([email protected]; correspondence for *Authors Israel. 69978, Tel-Aviv University, Aviv Tel Science, esi .Tillmann D. Kerstin proliferation links dynamics secretion and and levels Sec16 of Regulation ARTICLE RESEARCH ß 670 2014 December 10 Accepted 2014; May 23 Received sites cargo exit nascent ER shuttle called and 1 form ER vesicles the rough COPII the regulate where (ERES), of factors regions and growth ribosome-free known growth pathway. like secretory is early as cues little the of addition, such organization external functional In processes how fragmentary. cellular about still cell a membrane is traffic, other or of proliferation membrane integration polarity with of the cell field traffic of the orchestrate understanding in that comprehensive contrast, those By past as regulatory proliferation. of the number such in a of research circuits, understanding Intense our fostered biology. has developmental decades challenge and major cell transcriptional, a is in to networks respect post-translational and with translational homeostasis cell Understanding INTRODUCTION Signaling and Sec16, sites, secretion exit ER between WORDS: KEY link proposed previously linking mechanistic proliferation. integrator the a an provides for as This on secretion. basis acts dynamics and protein signaling its this factor and growth that levels propose Sec16A we regulate ERES, factors finding growth our we with that Together Finally, proliferation. whereas ERES. increases proliferation, overexpression at treatment reduces its Sec16A depletion factor of Sec16A growth that turnover demonstrate increased short-term to by leads regulated that also notion, this is with Consistent number. Sec16A growth ERES persistent increase detects to that stimuli loop factor central feed-forward family a coherent is Egr as a acts on in that Sec16A node dependent signaling. that protein manner hypothesize short-lived factor We a factors. in a growth transcription factors is of growth Sec16A by exit integrator that regulated reticulum an find endoplasmic we possibility as of Surprisingly, the acts constituent explore (ERES), major we sites Here, a homeostatic the growth. Sec16A, other cell of that with as understanding pathway such secretory mechanistic processes early broader the of a integration lack currently We ABSTRACT es Farhan Hesso nvriyo aruh aruh940 Germany. 95440, Bayreuth Bayreuth, of University Universita akA Hauser A. Mark itcnlg nttt hra BT)a h nvriyo Konstanz, of University the at (BITg) Thurgau Institute Biotechnology 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,6062doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. xotfo h nolsi eiuu E)ocr on occurs (ER) reticulum endoplasmic the from Export ¨ stas 0 osaz744 Germany. 78464, Konstanz 10, tsstrasse 1,2, 1 ro Mazza Arnon , * 1,2 eoiaReiterer Veronika , 4 i Atias Nir , 4 lvti colo Computer of School Blavatnik 3 xeietlPyisI, Physics Experimental 2 nvriyo Konstanz, of University 1 rnec Baschieri Francesco , 4 ailF Legler F. Daniel , ato oeetfe-owr op hc nertssecretion integrates which signaling. factor loop, growth feed-forward and as coherent and acts a Sec16 that proliferation of propose We part dynamics. growth, and levels cell protein Sec16 2011). controlling al., genes in et Fowler responsive role 1990; (Sukhatme, early a differentiation known Being play are members MAPK genes. family to Egr by signals, target mitogenic of induced in of downstream sequence h) consensus promoter (1–2 GCGG/TGGGCG a the rapidly to basal bind are low and a signaling but the exhibit of members, level, Proteins four expression comprises to pathway. which signaling known family, Ras–MAPK Egr are stimuli the several that induce of that downstream family protein responses factor transcriptional of mediate transcription (Egr) regulation response pathway. for gain secretory how relevant early to is the factors) of and need transcription the organization export by the we of whether instance ER level, (for regulation the abundance on post-translational the of the of regulation knowledge view at the to integrative Besides machinery important and pathway. are COPII full questions secretory involve these it a factor to does Sec16 obtain growth answers or by Sec16 with The What on ERES components? ERES? associated contingent of in solely are biogenesis changes signaling the changes to Is of lead molecular phosphorylation? phosphorylation 2010) and al., does dynamic et how (Farhan functional unanswered: Sec16 Several and established. remain details be to molecular questions remain regulation the this to al., but of 2013; reported consequences et export, were (Jin al., 2004), as ER ubiquitylation al., et 2011), et modulate al., (Dudognon such Koreishi glycosylation et or Sharpe 2010; 2012) 2011; modifications, al., al., al., et et et Lord post-translational Kung 2008; (Farhan 2010; al., Schwartz, phosphorylation 1995; Various et and al., 2007; et Whittle Ivan Espenshade 1996; 2012). 2008; 2006; Glick, al., al., al., et et et Watson and Gimeno 2002; ERES Farhan al., et 2005; (Bhattacharyya in Supek al., and role function et components COPII Connerly important their several an with interacts regulates plays it as Sec16) Sec16A homeostasis 2012). (Budnik al., called et pathway Zanetti secretory 2004; (hereafter al., the et Lee of Stephens,2009; sites and downstream to proteins eu-tre eaclsfr6h hc eutdi robust a in resulted which h, 6 we of for Therefore, response growth Sec16. cells the requires integrating HeLa depletion whether in serum-starved factor Sec16 determined growth of to first ERES role we the signaling, determine factor to ERES of order level the In at signaling factor growth integrates Sec16 RESULTS ntecretwr,w hwta rwhfcosmodulate factors growth that show we work, current the In growth early the of members are latter the for Candidates 1 oe Sharan Roded , 1,2 ui Hoffmann Julia , 4 atisWeiss Matthias , 3 aetn Millarte Valentina , 3, and * 1,2 ,

Journal of Cell Science eueEE ubr(ahne l,21) ncdw cells Knockdown 2010). al., et average on (Farhan to displayed number identified previously ERES we which reduce PIP5K1C), (NME5, and kinases NME7 Therefore, four NME6, levels. with experiments factor the knockdown growth performed renders in we the number changes ERES Sec31 to reduces in unresponsive that question cells depends condition the by reduction raises causally any observation whether starvation this the of However, (labeled Sec16 factor 1A). that growth (Fig. Sec16 ERES by on of showing ERES of of response, number knockdown this number inhibited Importantly, the immunofluorescence). in reduction ARTICLE RESEARCH 5 euto nEE ubrbtthe but number ERES in reduction ,25% atr splmnaymtra i.S) iia owa we what to growth similar of S1), amount COPII Fig. the of to the material number a insensitive (supplementary the initiates them factors reduced performed that rendered Sar1A and also GTPase of ERES Depletion status the We factor cascade. Sar1A, growth assembly S1A). in of Fig. changes knockdown to material respond to (supplementary the ERES affected knockdowns of four to these ability signal of factors none Sec16, growth through occurs levels. that ERES interpretation protein number the Sec16 with of ERES accordance independent in In is Fig. that reduction mechanism material a the (supplementary through that affected indicating not were S1A,B), Sec16 of levels ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal eut hwtemean the show Results 156.4 This to state. amounts steady value in a control as of presented percent cell per number ERES the of of representation graphic and knockdown a Sec16 of efficiency the demonstrating immunoblot an panel shows right The microscopy. confocal and by immunostaining followed Sec31 h fixation, serum 6 were for or (Strv) (SS) starved state were steady cells in h, left 72 After (siSec16). against Sec16 siRNA or (siControl) control siRNA with transfected were cells levels. regulate expression levels Sec16 factor Growth 1. Fig. el e xeiet * experiment. per cells with experiments independent ees h aaso h mean the show data bars) The (gray levels. Sec31 and bars) (black Sec16 on three experiments of independent evaluation an shows right panel the and a experiment shows representative panel left The proteins. indicated the for were immunoblotted lysates Cell times. the indicated for serum-starved or (NT) untreated 10 Student’s two-tailed proteins. indicated the for immunoblotted were FBS lysates to Cell prior stimulation. min 30 cycloheximide for CHX, treatment h. 4 10% for with FBS stimulated were or left untreated either were cells HeLa (D) condition. serum-starved the of percentage the show data mean The bars). and (gray bars) Sec31 (black Sec16 of expression depicting three experiments of independent evaluation an shows right panel the and a experiment shows representative panel left The indicated proteins. the for immunoblotted lysates Cell were indicated. as treated FBS or 10% h with 4 for cells serum-starved HeLa were (C) control. of untreated percentage the a as represented are and m .()HL el eeete left either were cells HeLa (B) m. 6 ..adaepeetda a as presented are and s.d. ts) cl bar: Scale t-test). P 6 6 , ..fo three from s.d. 2ERES. 32 .1(paired 0.01 A HeLa (A) . 6 50 s.d. 671

Journal of Cell Science rvne e1 nuto pnsrmtetet Egr treatment. nevertheless serum they upon levels, or Sec16 Egr1 induction cells reduce of knockdown se Sec16 knockdowns Egr3 per single not prevented or although did Egr1 note, Egr3 Of in response this a 3D). ablated in and (Fig. resulted expression, completely h protein 4 for Sec16 was serum on of with dependent the induction cells is whether of robust treatment Treatment determined mitogen Egr3. or we by Egr1 levels Next, Sec16 reduction 3B,C). of clear (Fig. shown) induction a levels in not resulted (data Sec16 Egr3 expression in and of Sec16 Egr1 knockdown reduce of co-knockdown not Individual but did and 3A). Egr3 (Fig. EGFR or candidates as Egr1 namely factors likely transcription receptors, most family Egr the factor revealed interaction approach growth protein–protein promoter This a IGFR. two Sec16 (in the distance to their in of network) sites sum binding the potential by factor and of based transcription number ranked were the of factors on transcription These presence This S1). revealed Table gene. the region material which Sec16 for 500-bp sites, the of scanned a binding site was analyzed start transcription bioinformatically region the we of this, upstream that test is levels. possibility as To Sec16 Another control granules 2013). factors structures al., transcription stress et such growth-factor-sensitive Buchan labels of shown; which not not formation VCP, (data for did the staining we for and using determined trapped evidence However, is any translation. it where observe undergoing granules, stress from in tested prevented mRNA next the of we storage synthesis. Thus, Sec16 degradation. regulates signaling proteasomal factor of growth increase whether an rate by in caused the decay not case the are in proteasome, starvation the serum the under by levels degradation mediated not Sec16 principle the in although was is that, Sec16 This conclude of the we higher treatment. Therefore, for are 2B). starvation cycloheximide (Fig. cause serum under under might main we kinetics than then the decay starvation, the is serum that under expect degradation levels cycloheximide-treated Sec16 increased in in reduction an decay proteasome, increased its If the MG132 prevented determined with cells. by and treatment we that degraded levels First, given Sec16 principle, case, both. the in of was which is, levels combination of Sec16 a rate Sec16 the either whether in by explained reduction in be a or by can decrease degradation, synthesis levels protein in Sec16 a increase in an by decrease in The 2B). resulted (Fig. of with half-life likewise translation a blocking starvation has treated after Sec16 levels that Sec16 cells revealed chasing Indeed, in protein. by controlled absent are levels signaling. was on Sec16 factor Thus, growth dependent it 1D). (Fig. was largely cycloheximide because with effect remained after treatment synthesis This levels levels (i.e. 1C). Sec16 Sec31 factors (Fig. increased growth contrast, back serum) and 2– By about Adding cells of 1B). halftime serum-starved unchanged. (Fig. a we with levels h Sec16, Sec16 3 in of reduction levels ERES a alters observed the factors growth changing of absence by the whether determine To decreases synthesis signaling Sec16 factor growth of Absence and Sec16 of presence the required factors ERES COPII. of growth ability to the respond Therefore, to cells. Sec16-depleted in observed ARTICLE RESEARCH 672 oto fSc6snhsscud npicpe cu through occur principle, in could, synthesis Sec16 of Control short-lived a is Sec16 that imply above shown results The 0cniae (supplementary candidates .90 – Fg A.Serum 2A). (Fig. h ,2–3 – fstimulation of h ,3–4 enovo de protein vlaino he needn xeiet eitn h xrsinof expression mean an the the shows show depicting Values panel experiments Sec16. lower independent the three and of upper experiment evaluation The representative to proteins. a subjected indicated shows were the panel serum- lysates against to the immunoblotting prior and and min lysed, SDS-PAGE 30 were for Cells times. MG132 (+MG132). indicated with starvation were the pre-treated cells for were HeLa serum-starved cells (B) were Alternatively, control. or untreated untreated the left of either mean percentage the a show as evaluation Values presented an experiments. shows independent panel panel lower three upper the of The and were experiment proteins. representative Cells indicated a (+MG132). the shows addition against cycloheximide immunoblotted to and with prior lysed pre-treated min were 30 cells for Alternatively, proteasomal MG132 indicated. by as rescued (CHX) is cycloheximide that protein short-lived a inhibition. is Sec16 2. Fig. h nrae control. untreated the A eaclswr ihrlf nrae N)o eetetdwith treated were or (NT) untreated left either were cells HeLa (A) ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal 6 ..adaerpeetda ecnaeof percentage a as represented are and s.d. 6 ..adare and s.d.

Journal of Cell Science EERHARTICLE RESEARCH iaerdcdSc6lvl Fg 3E). this (Fig. (also of levels depletion ERK2 Sec16 accordingly, reduced of and kinase signaling, downstream MAPK1) activated as known are factors transcription 244.25 ERES to microscopy. confocal amounts using value were imaged this cells and and non- HeLa Sec31 control with (F) for the transfected proteins. stained of were indicated fixed, percentage cells the were a HeLa for cells as (E) h, immunoblotted were indicated. presented 72 and cells as are After h, h immunoblotting results Egr3. 72 72 and and The and mean After after lysed lysis ImageJ. Egr1 Egr3. lysed were to using against or were cells prior counted siRNA Egr1 Cells h h, or were against (ERK2-KD). 72 4 siRNA siRNA ERK2 for After control with against (FBS) (Egr1+3). with or siRNA FBS Egr3 transfected siRNA with 10% and non-targeting were or with with Egr1 cells treated siRNA transfected against transcription or HeLa targeting the were siRNA which (NT) (B,C) cells of to directly HeLa interactions. combinations receptors harvested (D) physical factor different either proteins. documented growth two indicated two experimentally with the the show or are for Lines nodes (Control) immunoblotted Red nodes). siRNA promoter. (purple non-targeting Sec16 proteins the with to intermediate transfected bind through to linked predicted were factors transcription factors expression. are Sec16 nodes regulate Green factors text. transcription the family Egr 3. Fig. eesdb digboi,adclswr ie tteidctdsbeun iepit.Telwrpnlsosabrgaho loecneitniya h Golgi the at intensity at fluorescence intensity of fluorescence graph bar over a increase shows fold panel was the lower ManII–GFP h, The as time-points. 72 presented subsequent After fluorescence, indicated Egr3. 9.27 and the ER Egr1 at to against fixed siRNA normalized were or cells area, siRNA and control biotin, with adding transfected by were released construct RUSH mannosidaseII GFP-tagged expressing stably § 0clspreprmn.Saebr:10 bars: Scale experiment. per cells 50 6 6 r12.32 or 3 ..fo he needn xeiet where experiments independent three from s.d. 6 . ncoe 1ad# nEr+ ncdw el,rsetvl.Terslsso h mean the show results The respectively. cells, knockdown Egr1+3 in #2 and #1 clones in 2.6 m m. § 0clswr vlae e xeiet * experiment. per evaluated were cells 50 A ceai ersnaino h eut forbonomtcaayi sdsrbdin described as analysis bioinformatic our of results the of representation Schematic (A) fEE Fg F n h xeto hsrdcinwas reduction this of extent the and number 3F) the (Fig. in reduction a ERES in resulted Egr3 of and Egr1 of depletion nacrac ihteraiiyt euaeSc6lvl,co- levels, Sec16 regulate to ability their with accordance In ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal P .Ti au mut o9.52 to amounts value This t0. , .5(NV ihTkysps o et.()HL cells HeLa (G) test). hoc post Tukey’s with (ANOVA 0.05 6 ..fo he needn xeiet with experiments independent three from s.d. 6 36 RS h eut hwthe show results The ERES. 13.62 6 . ncnrlclsand cells control in 3.6 673

Journal of Cell Science n g3dul ncdw erae h ubro ERES of number line the Egr1 cell decreased cells. hepatic knockdown HeLa than the double cell of used a Egr3 secretory synthesis stronger to we the and a due this, in is alterations is which test to HepG2, cells To due of knockdown proteins. or number double levels secretory the Sec16 Egr3 in and in we change Egr1 reduction therefore and in observed 2006), ERES the Linstedt, and whether Guo determined 2008; al., et excluding (Farhan thus Sec16 pathway. S2B), secretory of the Fig. on effects depleted material pleiotropic Egr3 was trafficking (supplementary cells and membrane Egr1 was plasma observed to of in effect Golgi effect on ManII- the No knockdown observed and of double S2A). 3G), arrival Fig. (Fig. that the material Golgi (supplementary in the to delay to marked ER comparable a the of to GFP- from knockdown led expressing RUSH Double Egr3 stably and (ManII-RUSH). cells Egr1 enters II used that mannosidase We cargo tagged the pathway. liberates cells secretory ER– and the the retention on of relieves effects Treatment biotin secretory for retention. with of (RUSH) retention the streptavidin-based test ER using on to used hooks cargo relies 2012) system We selective This al., trafficking. 2010). Sec16 Golgi using et al., in (Boncompain retention et observed system Farhan previously described 1; have recently (Fig. we cells what knockdown to comparable ARTICLE RESEARCH 674 ag odhspeiul ensont fetEE number ERES affect to shown been previously has load Cargo n g3dul ncdw eue ertr ag load. cargo Egr1 secretory that reduces prevalent unlikely knockdown is most double it the Egr3 Therefore, 4C). and albumin, (Fig. with hepatocytes obtained 2010). in al., cargo was et Reiterer result 2008; similar al., the et A affect (Nyfeler cells not HepG2 did in protein Egr3 and of Egr1 levels silencing However, 4A). (Fig. atr n e1) h oncinogtt econsiderably fast be a to of ought the connection presence a trigger growth The between the Only Sec16). to (i.e. node and necessitates able extent. central factors and CFFL is appreciable input between A stimulus any connection persistent) node. not to do a output node node central (i.e. the output prolonged trigger to the able that affect are ensure which that are detectors, CFFLs stimuli persistence number). transient of ERES part as or found (secretion typically node subsequently node that output input (Sec16) an an node CFFL, triggers central a 5A) a In Fig. triggers 2002). factors) al., (CFFL; et (growth loop Shen-Orr 2013; feed-forward al., coherent et (Lim a of scenario This reminiscent load. is In cargo induce secretory stimulation. to increasing factor expected thereby growth are translation, signaling after pathways Sec16 signaling h factor in factor 2–3 growth increase growth of addition, timescale an that a in on revealed resulting levels have transcription, Sec16 far regulates loop so feed-forward results coherent a Our of part as Sec16 ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal a nirpi AT)(i.4) ao secretory major a 4B), (Fig. (AAT1) antitrypsin 1 aine hsvleaonsto 314 amounts value This inter-assay variance. for account the to of control percentage are a results as The presented cell. per ERES number of the of a representation shows graphic panel left The confocal microscopy. by acquired and were Sec31, images for were stained cells and h, fixed 72 After and (Egr1+3). Egr1 Egr3 against siRNA or (control) siRNA control with transfected in cells. load HepG2 cargo of Egr3 independent and Egr1 is of loss ERES after in number decrease The 4. Fig. mean cl a:10 bar: test). Scale hoc post Tukey’s with (ANOVA * experiment. per evaluated where experiments mean the show Values AAT1. glycosylated core arrowhead, AAT1; glycosylated complex Arrow, independent experiments. three of evaluation panels show right the and experiments left The representative C. in show albumin panels for or B in (AAT) and lysed were for cells immunoblotted h, 72 After and Egr1 Egr3. against siRNA with or siRNA non-targeting with transfected were ecnaeo h control. the of percentage 6 6 6 1EE.Terslsso the show results The ERES. 51 ..fo he independent three from s.d. ..adaerpeetda a as represented are and s.d. m A eG el were cells HepG2 (A) .(,)Hp2cells HepG2 (B,C) m. a § 1-antitrypsin 0clswere cells 50 P , 0.05

Journal of Cell Science EERHARTICLE RESEARCH rvosyrpre iuainapoc o h efasml of a self-assembly adapted the we for number, approach ERES simulation in reported previously increase 1). observed (Fig. be ERK2 the treatment of underlies to min on did 30 unlikely cells levels first dependent Sec16 the is because Sec16-depleted in levels, change and was Sec16 not in of S1D,E) induction response case by Fig. mediated rapid the material This (supplementary not with ERES was 5B). them in (Fig. increase treated ERES rapid which a min) in observed (10–15 number, We decrease (FBS). briefly serum a bovine and in fetal 1A), results (Fig. which number cells, serum-starved We alters and number dynamics ERES Sec16 increases treatment brief factor organization. Growth a ERES how and and Sec16 whether affected tested growth treatment next between factor (i.e. We growth nodes secretion). output and and factors input between than faster results a The shows variance. panel interassay right for account The microscopy. to (Strv) confocal control by of imaged or percentage fixed, and a and Sec31, h as for 6 presented stained for are (Strv) were Results starved Cells cell. serum per fixation. were mean cells ERES and the h, of (FBS) show 72 number min After the 15 (siSec16). of Sec16 for representation against treatment graphic siRNA FBS or (siControl) 10% siRNA by factors. control followed growth with transfected to were Sec16 cells HeLa of (B) response Fast 5. Fig. rwn e RS D eaclswr eu tre o Sr)adsiuae ih1%FSfr1 i FS olwdb iainimnsann and 96.9 immunostaining panel to fixation lower amounts by The value followed analysis. This for (FBS) Strv. appearing min to as compared 15 thereby particles change for Sec16, counted fold FBS dissociatio of more the 10% cells, masks as even with as mitogen-treated presented attract stimulated well size In that 462 and as ERES arrows). membranes or ERES (Strv) of (red of ER h representation ERES images on 4 graphic representative existing a for assemblies shows shows by starved local panel captured serum form upper were that is can The Sec16 cells microscopy. it molecules arrows). confocal HeLa where dashed Sec16 (D) black membranes, rebinding (thin ERES. ER and ERES new on the arrows), growing leaves diffuse dashed rarely and black Sec16 to cells, (thick starved rebind enhanced In will modeling. mathematical ERES our the of escapes results the of representation Schematic (C) curdeey6 eod.Sil r hw fteidctdtm-ons h ih aesaemgiidaesadtenmest h ih r h enERES mean the are right the to 10 numbers bars: the Scale and region. areas magnified displayed are the panels within right The number time-points. their indicated and the pixels) of shown (in are size Stills seconds. 60 every acquired Student’s ogi oedtie nihsit h ehns that mechanism the into insights detailed more gain To 6 6 mfrec RS.Terslsso h mean the show results The ERES). each for nm 462 ts) E eaclsepesn F–e3Awr eu tre o .Lv mgn a tre pnadto f1%FSada mg was image an and FBS 10% of addition upon started was imaging Live h. 4 for starved serum were YFP–Sec31A expressing cells HeLa (E) t-test). 6 ..fo he needn xeiet where experiments independent three from s.d. A ceai ersnaino h rpsdchrn edfradlo.Pop. phosphorylation. Phosph., loop. feed-forward coherent proposed the of representation Schematic (A) 6 ..fo he needn xeiet with experiments independent three from s.d. § 0clswr vlae e xeiet * experiment. per evaluated were cells 50 m m. ucino h rti density, protein the of function rtisaddfuincefcet( coefficient diffusion and proteins hsapc loe st nltclypeittesteady-state the predict analytically ( ERES to of radius us and number allowed on include aspect to rate times Flory–Huggins scenario with this Flory–Huggins residence average the standard on Extending finite membranes. dissociate ER a they have i.e. to COPII membranes, ER and/or contrast Sec16 in scenario, (the membranes However, ER on ERES). domains a patchy show into COPII segregation and/or a Sec16 dynamic of water, formation in domain polymers and Flory– a amphipathic the to small of Analogous size scenario 1995). ER unmixing (Gedde, formation type an on uniform Huggins by ERES proteins described of be state fairly quasi-crystalline COPII can steady therefore or The a Sec16 ERES. a between of with membranes density revealed low experimental ERES comparatively with simulation of agreement In arrangement our 2008). al., observations, et (Heinzer ERES ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal § P , 0clspreprmn.* experiment. per cells 10 .1(ardtotie Student’s two-tailed (paired 0.01 N ERES w n h iscainrt fthe of rate dissociation the and , 6 92pxl/RS( pixels/ERES 19.2 and C R ERES and epciey sa as respectively) , D P , respectively): , .5(paired 0.05 , 9.8 6 t-test). . pixels 9.8 C from 675 nis

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Journal of Cell Science iscainconstants, dissociation iecl fteFA xeiet(eaieaon 1 of amount is the rate (relative population dissociation on Sec16 experiment averaged entire recovery the arithmetically fluorescence FRAP the the the Consequently, to of contribute timescale carries not that does pool Sec16 that amount fast (relative a recovery observed of the of existence the value requires maximum therefore a with recovery hra h aftm ftercvr sol eemndby determined only is recovery the of time half the whereas ( obe fe ioe ramn Fg G.Ms iey the likely, Most which 6G). fraction, (Fig. mobile treatment the in in mitogen change increase after the than of doubled range smaller the thus in was is treatment mitogen upon diffusion oprdwt ioe-rae el ( cells mitogen-treated with compared otlkl en httefs iscaingismr egt( weight more gains that dissociation Given fast the increases). that means likely most nafse unvrkntc fSc6o vrg ( average on Sec16 of kinetics turnover faster a on in increase an EERHARTICLE RESEARCH soito fCPIwt RS(ahne l,20;Forster the 2008; concomitantly, al., and, et ER (Farhan ERES the with in COPII load cycloheximide of protein with association cells the ERES treated reduce we to step, to COPII recovery, first fluorescence of a of As binding amount experiments. the the that new affect decreasing generate and to to then ought pool Sec16 true, of Sec16 ability slow If the a in ERES. role and a fast plays COPII a thereby for of responsible existence is observed COPII with the interaction an that hypothesized We Sec16 of turnover ERES the on modulates COPII with Interaction 1/ than time less much takes experiment the (because fmnts ossetwt tbigpr faCF.W next We CFFL. generates a Sec16 of of patches part mobilization ERES. the ER more being how it understanding on at with copies aimed timescale consistent the Sec16 minutes, on regulated of of is Sec16 diffusion Nevertheless, ERES. mean between consequently the which structures, reduces a oligomeric in our of pronounced resulting formation more e.g. of transient components, a ER-resident to of timescale other leads pool with possibly the interaction the molecules Increasing Sec16 ERES. on free Sec16 between these of regions (immobile ER amount on the ERES molecules increases in ‘mobilization’ this stuck experiments), is that hoeia rdcin o oul rti of with protein ( well soluble treatment agreed the a cytoplasm of diffusion the for irrespective the in predictions that found GFP–Sec16 theoretical We in of shown S3). are constant Fig. curves FCS material cytoplasm (representative supplementary in membranes GFP–Sec16 ER of on constants and diffusion us the allowed determine that to (FCS) spectroscopy we correlation this, investigate fluorescence To used cells. mitogen-treated and true, starved membranes, hold between ER to is on rationale Sec16 of constant that factor ERES. a that new is conclude of stimulation formation therefore mitogen drive rate we can after and dissociation Sec16 model, of mean above mobilization the the of in prediction increase an ol xetete ulrcvr ( recovery full a either expect would opeeyutetdcls( cells untreated completely ebaebudpo fSc6soe euto nthe in ( reduction starvation a serum showed under Sec16 constant of diffusion pool membrane-bound q )o h iecl fteFA xeiet bevn a Observing experiment. FRAP the of timescale the on 50) h ubradsz fEE lodpn ntediffusion the on depend also ERES of size and number The fol igepo fSc6GPwspeeto RS one ERES, on present was Sec16–GFP of pool single a only If D n in and q fe rwhfco tmlto hrfr reports therefore stimulation factor growth after C r linked: are C C slow 5q D utvr oalse xetthan extent lesser a to vary must C sascae ihapo fSec16 of pool a with associated is D fast C <0.62 D C 5q +(1 <16 stewihe vrg ftwo of average weighted the is C 2q q fast q m )o orcvr tall at recovery no or 51) m q ) m ewe eoadunity and zero between +(1 ,adaso e1 pool Sec16 slow a and ), m C D 2 slow 2 s.Ti nraein increase This /s). hs forabove our if Thus, . s.B otat the contrast, By /s). D 2q < ..increasing i.e. , D C ) 0.65 < C C slow 0.43 seatythe exactly is sincreased), is m Observing . C q 8 kDa, ,280 m 0 and ,50% nFRAP in , m slow 2 m s and /s) 2 .Yet, ). s as /s) 2q C fast C C ). q motn o h eeaino e RSatrgot factor be growth after might ERES interplay new COPII–Sec16 of generation the treatment. the that for the propose important inhibit 6F,G). (Fig. we not 5B). treatment did EGF Fig. Thus, treatment after also mobility factor not GFP–Sec16 and growth 1A; in did S1A) to (Fig. increase but respond Fig. to ERES, material treatment ERES of (supplementary of number new ability factor the form the lowered affect to NME6 growth ability of the Depletion Of affected upon 6E,G). also (Fig. EGF Sar1A ERES found mobility of and GFP–Sec16 Sar1A depletion the change depleted note, support we not COPII, further did of To treatment role mobile not the 6D,G). and of (Fig. recovery did notion of Sec16-Maddin factors extent 1997). of previously growth the al., fraction decreased with et been rather (Shaywitz treatment but has Sec16 increase experiments, of Sec31 region FRAP region with central Sec31-binding interact In the to the able to Sec12 still mapped as and is but 6H) Sec23A Sec23A, (Fig. fails Using with to Sec16-Maddin 2012). interact that al., to bind confirmed et we to Montegna 2007; co-immunoprecipitation, described Glick, the lacks and was it (Bhattacharyya but Sec16- that 2010) called al., et ERK domain we (Farhan described Thr415 previously which the site acids, contains phosphorylation still lacks amino mutant performed that This 431 we Sec16 Maddin. C-terminal of COPII, mutant last of truncation role the a 6C,G). the with (Fig. into experiments cells of insight FRAP mitogen-treated extent further and cells obtain starved the cycloheximide-treated To for recovery, in same fraction fluorescence the mobile was Sec16 the in and led recovery increase cells control in an treatment factor to growth Although 2006). al., et fSc6wt OI srqie o h omto fnwERES new of formation Sec16 the for of treatment. required mitogen necessity is after COPII cooperation the with 7B–D). that Sec16 highlight (Fig. underscore of and generation, ERES data ERES for COPII-positive our mobilization and their and together, reduced Sec16 Sec16- Taken from significantly and of by COPII cargo COPII cycloheximide formation secretory of of with and load the fresh recruitment treatment Reducing ERES. Sec16 with to new of incubation led formation of by which followed majority cytosol, 7B), the (Fig. to endomembranes washed off extensively were the strip cells Semi-intact for assay. treatment. relevant mitogen recruitment upon be GFP–Sec16-Maddin ERES to of with number seems the cells in Sec16–Sec23 increase increase not in Hence, the did also 7A). stimulation number whether (Fig. ERES found mitogen 7A). tested (Fig. and case after we Thr415 the of be control, to phosphorylation this a Sec16 required wild-type As with response 7A). factor labeled (Fig. ERES growth of GFP–Sec16 Sec16, number the endogenous increased with treatment factor more observed growth of to As generation respond to the treatment. of fail to ability should crucial Sec16-Maddin the GFP–Sec16 is then If COPII ERES, of imaging. with confocal interact avoid variability by with to followed Sec16 treatment high to min after 15 too and intensities for before serum a fixed fluorescence were with Cells of overexpression. cells range of twofold inclusion cells a compared only within ERES We Sec23- the or GFP–Sec16-Maddin. GFP–Sec16 determined wild-type binding-deficient either we expressing of treatment, cells generation in mitogen the number in after interplay ERES Sec16–COPII the more of role the test generate To to Sec16 for ERES required more is COPII with Interaction ial,w etdterl fCPIuigan using COPII of role the tested we Finally, ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal enovo de nvitro in 677

Journal of Cell Science EERHARTICLE RESEARCH xotadcl rlfrto.T etti xeietly we 678 cell experimentally, in this increase the test measured were and ER To knockdown Sec16 between proliferation. that a link performed cell a processes and suggesting 8A), export (Fig. proliferation-related enriched of significantly several approach enrichment this Indeed, for annotation). revealed analyzed term (GO was al., processes network et biological (Yosef anchored therefore as previously This Sec16 described We with algorithm 2011). hits an proliferation. 111 using these anchor, a cell of an then network in to anchored enriched proliferation, an be related ER constructed to cell ought if processes of Sec16 to that, regulation cellular (111) hits the hypothesized these to linking We network ERES linked 2012). regulate is al., that export et when proteins (Simpson 47 that, More previous 2010). described number of phosphatases al., have our et and (Farhan others outcomes ERES In recently, kinases of biological number the 64 proliferation. reduce the depleted, identified as unclear mitogenic for we is such work, it integrates relevant far, signaling, Sec16 thus is mitogenic However, that ERES. Sec16 of indicate whether level far the at so signaling results Sec16 on Our dependent is proliferation Cell esta el xrsigwl-yeSc6 e1 forms Sec16 with Sec16. oligomerize to mutants wild-type on expected are expressing mutants effect these significantly Sec16 cells and the but oligomers overexpressing cells, than determined GFP-expressing less cells than and more Sec16-Maddin proliferated HeLa as proliferation. mutant well Sec16-T415I the as overexpressed sufficient also and We necessary proliferation. is Sec16 for that Overexpressing indicating S4), supplementary Fig. 8E). 8E; material (Fig. (Fig. Egr3 proliferation Egr- Sec16 induced was significantly and the which Sec16 overexpressing proliferation, in Egr1 cell by of Sec16 involved Silencing inhibition overcome an in is proliferation. in resulted Sec16 increase of together whether control threefold tested dependent a We Ras 8D). levels, induced Egr (Fig. Sec16 control induce also members to Egr In overexpression that 8D). known finding (Fig. our also verified with experimentally is line caspase-3 we well-known it which determining a members, and family is by proliferation, pathway of Ras–ERK1/2 assessed regulator The as 8C). (Fig. an inhibited apoptosis to cleavage Sec16 attributed not of in was effect depletion this increase that and 8B), found (Fig. We proliferation cell days. 2 for number ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal wse’ ausso h mean the show condition Values the ‘washed’. to normalized the ‘+cytosol’ Sec16- under condition in ERES increase Sec31-positive relative and the positive bar shows the B and in ImageJ, graph using counted were Sec31-positive ERES and Sec16-positive of The numbers immunofluorescence. Sec31 using and detected room Sec16 were at fixation. min by 30 followed for temperature (+cytosol) harvested cells cytosol HeLa with from after treated directly or fixed (washed) either washing were and cells COPII These deplete Sec16. to permeabilized extensively were washed h Cells and 2 experiment. for the (CHX) to not cycloheximide prior either with were treated cells or on h, treated plated 24 were After cells coverslips. HeLa glass (B–D) FBS-treated lines). or (white cell lines) cells per (black ERES cells of non-treated number from median the ImageJ. indicate of using Lines number counted The was fixation. ERES for by Sec16-positive FBS followed 10% (+FBS) with min treated 15 either or were (NT) cells directly h, fixed 24 After (T415I). GFP– Sec16-T415I or (Maddin) GFP–Sec16-Maddin GFP– (WT), wild-type Sec16 encoding plasmid with biogenesis transfected the in Sec16 ERES. and COPII of of Role 7. Fig. Sc6 rtre(e3)idpnetexperiments. independent * (Sec31) three or (Sec16) P , .5(ardStudent’s (paired 0.05 A eaclso ls oesiswere coverslips glass on cells HeLa (A) t ts) cl as 10 bars: Scale -test). 6 ..fo four from s.d. m m.

Journal of Cell Science EERHARTICLE RESEARCH vrxrsino a1 a odtcal feton we effect and linked, detectable are However, proliferation cell 8B). no the and (Fig. support export findings ER had our proliferation that together, notion Taken Sar1A 8E). reduced and (Fig. Sar1A proliferation of it depleted we overexpression similarly, that behave regulator would any found export whether test tolerate ER To not plasmid. of did any of cells of depleted overexpression silenced cells the Sec16 in variants because Sec16 Sec16 of although endogenous overexpression could proliferation, the We test Sec16. induce not wild-type overexpressing reason themselves than the less is mutants significantly this these that propose why We Sec16. wild-type endogenous rps htSc6i h oeua nertro hs two these of integrator molecular the is processes. Sec16 that propose tmlto nrae RSnme,btde o fetthe affect not does but increasing factor number, for growth ERES detector Transient a increases signaling. persistence of mitogen stimulation a part upon is as flux Sec16 secretory functions that propose that current We growth CFFL ERES. the of integrator into the an In signaling as into acts factor understood. Sec16 networks how poorly demonstrated signaling we work, remains and pathway metabolic secretory of integration The DISCUSSION ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal ue’ othctest). post-hoc Tukey’s the show and (t0) mean 0 time-point that at with plated compared the cells in of increase number fold the as are presented results The a chamber. using counting counted cells and transfection, detached after were h 48 At Sar1A. or Sec16-T415I phosphorylation-deficient the (Maddin), Sec16-Maddin wt), (Sec16- GFP–Sec16 wild-type GFP (GFP), encoding plasmid were with cells transfected h, 24 Egr3 After and (siEgr1+3). Egr1 against siRNA siRNA or control with transfected were cells HeLa (E) experiments. independent from three blots representative are Shown proteins. indicated the for and immunoblotted lysed after were h cells 24 transfection, At (RasWT). Ras or wild-type (control) vector empty with transfected the as mean caspase-3 total versus of cleaved percentage the depicting independent experiments three of shows evaluation panel an lower the and representative experiment, a shows panel The upper caspase-3. for immunoblotted Lysates were overnight. (BFA) 5 A with brefeldin treated or siRNA Sec16, or against siRNA control with transfected the show results mean at The plated (t0). that 0 to time-point the relative in cells increase of fold number the as results presented The are chamber. counting counted a and detached using were cells h h, 24 48 After or (siSar1A). Sar1A or Sec16 (siSec16) against siRNA or siRNA (siControl) control with transfected cells were and HeLa (B) growth displayed. cell are to proliferation linked are that enriched and significantly are Cellular that Cytoscape. processes in plugin BiNGO using the annotations) term cellular (GO of processes enrichment for that analyzed network were merged the all from shows nodes panel lower The (right network). merged and were Sec16 networks to the anchored also hits) were Pepperkok that and al., hits et (Farhan Simpson 2012) 2010; al., et screens (Farhan previous from hits shows proliferation. and cell pathway secretory link the a between represents Sec16 8. Fig. 6 6 6 ..* s.d. were cells HeLa (D) s.d. were cells HeLa (C) s.d. P , .5(NV with (ANOVA 0.05 A h pe panel upper The (A) m g/ml 679

Journal of Cell Science hssuyfcsdterl fmtgn nrgltn RS others ERES, in regulating identified in recently mitogens Although of have a role flux. e.g. the secretory fully cell, focused the study a of this remaining of of state capable emergence while steady COPII-dependent the is strict of response features components key cellular with mutual COPII compatible dynamic adjustable and a an Sec16 other of including Unlike model of 2012). our al., the stabilization et reducing interpretations, (Kung instance, would static Sar1 for of Sec16 more ERES, hydrolysis ERES, on GTP factor the of COPII rate stabilize to growth to recruited to act Once of then interaction respond COPII. factors an requires with growth not number) is ERES to Sec16 in did response increase to that rapid adaptive the Sec23 ERES Sec16-Maddin the (i.e. that of to that more indicates response bind fact treatment is adaptive to The situation an signaling. unable the of factor that context growth propose the we and in disassembly dynamic ERES, mitotic of of template context the this reassembly in Although correct dynamically. be how might regulated and model is mediated it is function whether template of and it this biogenesis However, how the components. unclear for COPII remains ‘template’ of at a independently cluster form acts to to that able and ERES is ER Sec16 the that ERES of al., proposed domains et with was (Hughes 2008). it COPII associated Therefore, of al., remains 2009). presence et the Sec16 without (Ivan mitosis that throughout co-factors shown from have help Others major without region (www.genecards.org). Sec16 miRNAs that al., these found et we by search, (Wang regulated database factors is our transcription to family according shown and, Egr been 2010) by have regulated miR-132 serum not be and to under by miR-212 did regulated instance, be formation we For could Sec16 miRNAs. granule However, that is storage possibility levels. Another starvation. for factor evidence growth promoter released and observe in direct stored be changes than could mRNA upon other Sec16 instance, also mechanisms For might control. through expression not Sec16 regulated did few promoter. transcription We Sec16 a be these the Egr3? whether after to to and test bind flux ERES to Egr1 factors Egr3) dissection secretory by rendering promoter higher regulated and a ERES, Sec16 a perform Egr1 is with of How deal on size hours. to are reduced dependent capable levels the fully manner Sec16 for load, a cargo compensate (in in scales. increase time increased longer an at secretory with size of appropriate the wave Concomitant an rapidly upcoming at acquire an is only with happen ERES they cope cargo, of to to number able modeling be the to Although our increasing size. by ERES in increase of predicted rapid expense is This timescale. number short a ERES on secretion) (i.e. output ARTICLE RESEARCH ned uhacnlso sfclttdb eetsre fthe of screen to 680 recent linked a cancer. by is facilitated as is expression such conclusion a proliferation Sec16 such cellular that Indeed, we deregulated this, speculate on of Based to conditions proliferate. to tempted cells of are ability the for relevant is levels. binary factor properly growth the to in that ERES alterations for show essential to also is respond COPII that with stress. findings manner Sec16 to cooperation our of response interaction to a COPII a the analogous in in illustrating is other components This Sec16, COPII form and and and Sec16 structures Sec16 between Sec24AB These on where stored. dependent bodies’, are the ‘Sec in components results of starvation acid is Amino formation which 2011). previously reported al., 2014), starvation et al., serum (Zacharogianni to et response the (Zacharogianni from starvation distinct acid amino to e1 a hw obn oEE hog narginine-rich an through ERES to bind to shown was Sec16 u idnsas niaeta e1 o erto ngeneral) in secretion (or Sec16 that indicate also findings Our Drosophila oe epneo ERES of response novel a odlse l,21;Smsne l,21;Ci ta. 2012). al., or et Chia 2012; 2010; al., al., metabolic secretory et et Farhan Simpson the 2006; 2011; signaling, al., on al., et et effect (Bard Kondylis levels numerous an various have at pathway that identified approaches regulators biology transcriptional have systems the recent on of that at view results aiming when integrative the important An deconvolving more secretion even proliferation. of becomes pathway and regulation secretory the and growth between treatment cell mitogen link and to of close response number a cellular the imply the therefore in of changes part downregulated that are al., ERES demonstrate et (Roeckel findings was cancer Our and ERGIC-53 2009). between ERGIC-53 link the on reports secretory as all of upregulation (Wis components, general to due pathway not samples (Wis be was tissue This cancer to healthy 2012). healthy in found colonic expression was its comparing in with Sec16 compared twofold patients tissue. approximately diseased cancer the upregulated with colon tissue eight colonic of proteome fe bleaching. after l xeiet eepromda 37 at performed a using were microscope experiments confocal LeicaSP5 All a with 63 performed was FRAP photobleaching after recovery Fluorescence METHODS AND MATERIALS ul 9 eedwlae rmEB n3 uut2012. 2006) al., August taken weight 2011). was et al., 30 network position et (Matys interaction (Yosef protein–protein ANAT on as TRANSFAC from A 11.1. from represented EMBL release downloaded database sites, from were Genome binding downloaded Human matrices on factor were based Transcription regions, from 19, the accordingly. network factors for build interaction transcription annotations the protein–protein and ranked a and Sequences transcription IGF1R in according or 180 factor EGFR distance transcription either to shortest enriched regions its each corresponding scored to 500-bp analysis matrices further This similar We genome. enriched factors. entire to the 184 for respect sites revealed start sites. 500-bp with transcription binding the of enriched calculated scan upstream for to site was 2003) start al., transcription Enrichment et the of (Elkon upstream algorithm region PRIMA the used We analysis sequence effects Regulatory possible overexpression. total intensity excluding GFP–Sec16 only of thereby fluorescence grade cells, were range), the comparable ERES to intensity GFP–Sec16-expressing due and twofold displaying a measured, of cells (within was image from cells case from quantified of originating the intensity noise from fluorescence excluded represented In were non-specific typically size exclude pixelization. in these pixels to as 2 images than analysis the smaller by Structures to ImageJ structures. using thresholding quantified uniform were LeicaSP5 applying ERES 63 a magnification. a using digital in threefold acquired or with Sec31 were microscope, or Images Sec16 confocal GFP–Sec16. for immunostained expressing cells cells in quantified were ERES quantification ERES fe recovery, after lahn,and bleaching, Srgee l,20) h oiefato a acltdas calculated was fraction mobile The reaction-dominated 2006). literature the the in al., used used q using widely et we analyzed been curves, were has (Sprague that Images images FRAP recovery second. bleaching, fitting single-exponential per After For ms. image 750 one ImageJ. was for mM at ERES 20 intensity the acquired image, and laser with were pre-bleach 100% GmbH) a supplemented at of Services acquisition (DMEM bleached After Imaging 7.4). medium (Life pH imaging HEPES chamber with Ludin covered a to transferred 5 6 ( 14N i-meso betv tfvfl iia magnification. digital fivefold at objective oil-immersion NA /1.4 F nesie l,21) hc si gemn ihearlier with agreement in is which 2012), al., et ´niewski ‘ 2 F 0 ora fCl cec 21)18 7–8 doi:10.1242/jcs.157115 670–682 128, (2015) Science Cell of Journal )/(F i 2 F F 0 F i 0 stefursec ntebece eindirectly region bleached the in fluorescence the is stefursec ntebece einbefore region bleached the in fluorescence the is ,where ), F ‘ 6 sfursec ntebece region bleached the in fluorescence is 14N i-meso betv at objective oil-immersion NA /1.4 ˚ .Gascvrlp were coverslips Glass C. nesie al., et ´niewski

Journal of Cell Science ta. 03;k 2003); al., et nt C aawr itduigteftigfnto o w non- two for function fitting the Airy one using to diffusion: fitted set normal was with were populations pinhole data interacting The nm). FCS (500–530 filter unit. bandpass a used h pia xs e wn oteuaodbeeogto fteconfocal the of elongation along unavoidable the diffusion by to (described captures owing volume factor yet This axis, summand. optical the first the of denominator C esrmnswr efre naLiaS5SDsystem SMD 37 SP5 for chamber Leica climate a custom-made on a performed with were equipped measurements FCS spectroscopy correlation Fluorescence ARTICLE RESEARCH upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.157115/-/DC1 online available material Supplementary material Supplementary RGY0076/2010]. number Frontier [grant Human Program and Science WE4335/2-1]; number Deutsche [grant Deutsch-Israelische 309/10-1]; Forschungsgemeinschaft the GA the from number of of support [grant Canton Fund the acknowledge Projektkooperation by Scholar M.W. and Young and Foundation; the J.H. Science by (SNF) German Thurgau. SNF; Foundation the by the Science Konstanz; from National of grant Swiss University R’Equipe the a from by grant and a by supported is H.F. analyses. Funding bioinformatic performed D.F.L. R.S. data. and analyzed N.A. and A.M., experiments V.R., reagents. performed manuscript. contributed M.A.H. the manuscript. and wrote the V.M. and wrote J.H., F.B., data and analyzed data experiments, analyzed study, performed K.D.T. the conceived H.F. and M.W. contributions Author interests. financial or competing no declare authors The interests Competing on discussions helpful for Zimmermann scenarios. W. and separation Brandt phase H. thank to like would M.W. Acknowledgements ohdfuintmsaedtrie yterdu ftecnoa volume, confocal the Sec16–GFP. of radius membrane-bound the by r e.g. determined are species, times diffusion diffusing Both slow a the amplitude describes whereas with Sec16–GFP, cytosolic term e.g. second species, diffusing fast a describes ebaepoen Mrzv ta. 01,fo hc n nesa D infers of one range gross which the from in 2011), Sec16 of al., constant et peripheral diffusion (Morozova for relation proteins Saffman–Delbruck membrane the employed we membranes, ipiiy ehv elce factor a neglected have we simplicity, nmmrnswr eie sflos easmdSc6t be to Sec16 hydrodynamic assumed a equation and Stokes We GFP than follows. larger 10-fold radius as about mass a derived with globular were obtained membranes the and text. on starved of main for the mean 16 in cells; the reported untreated are and cells) for treatments. mitogen-treated curves individually, and (24 cells away fitted of coefficients i.e. variety diffusion were ER, a in peripheral curves loci the several of FCS for regions rim, in nuclear s of the 60 from for timescales collected on were curves GFP 300 beyond of to well and photophysics were photophysics 20–100) times diffusion the typically all (here Because encodes focus also the in it molecules Sec16 GFP-tagged prefactor The here. reported eue yooi viscosity cytosolic a used We ape eeilmntda 8 mtruhawater-immersion a through nm 63 488 APO at PL (HCX illuminated objective were Samples 0 & h is emwt amplitude with term first The hoeia rdcin o ifso osat fSc6i yoo and cytosol in Sec16 of constants diffusion for predictions Theoretical 2n,adtersetv ifso osat,D constants, diffusion respective the and 220nm, , .-odlre hnGP i.e. GFP, than larger 2.2-fold B A D stemleeg.Frtedfuino e1 on Sec16 of diffusion the For energy. thermal is T ~ C a elgbefrteftigpoes Autocorrelation process. fitting the for negligible was ðÞ k t B S T ~ 2 < = (  5 thdltl nlec nteftparameters fit the on influence little had it 25) 6 1 pg z A ( 6 R 1 g fA t spootoa oteivrenme of number inverse the to proportional is { ) = -odlre hnta fwtr(Elsner water of that than larger 4-fold h ifso osati D is constant diffusion the .WCR) loecnedetection fluorescence CORR), 1.2W t f D ( 1 f ) ) n ifso time diffusion and n ifso time diffusion and R z 5 . m ae nteEinstein– the on Based nm. 3.3  1 ðÞ z 1 r { t  ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi = f t 1 D ( A m z 2 ) ,tecnrbto of contribution the s, m t 5 = ( 1 S c 2 m t ˚ t t D ( incubation. C m n D and D ( 2 D ( 1 c ) 2 ) 5 ~ 2 ) ~ ) /s. 17 r r 0 2 , 0 2 = m nthe in = m 10 ( ( For . 4D m 4D 2 m m /s. c s. ) ) lo,R,Lnat . hrn . hmr .adSio,Y. Shiloh, and R. Shamir, R., Sharan, C., S., P. Linhart, J. Levi, R., Paccaud, Elkon, E., and L. D. J. Strongin, Carpentier, C., A., Maeder-Garavaglia, P., E. Dudognon, Montegna, M., Esaki, L., P. Connerly, ha . o,G,Rcn,V,N,S,Kmr .adBr,F. Bard, and P. Kumar, S., Ng, V., Racine, G., Goh, J., Chia, sesae . ieo .E,Hlmce,E,Tug .adKie,C A. C. Kaiser, and P. Teung, E., Holzmacher, E., R. Gimeno, P., Espenshade, S. B. Glick, and D. Bhattacharyya, H., Kitayama, K., Saito, E., Wallace, A., Mallabiabarrena, L., Casano, F., Bard, References lnr . ahmt,H,Smsn .C,Cse,D,Nlsn .adWeiss, and T. Nilsson, D., Cassel, C., J. Simpson, H., Hashimoto, M., Elsner, J. D. Stephens, and A. Budnik, ose,R,Wis . imran . enu,E . eism,F., Verissimo, G., E. Reynaud, T., Zimmermann, M., Weiss, R., Forster, R., Sharan, Y., Silberberg, E., Fava, S., Mitrovic, W., M. 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