nml,pat n ug) pcmlxn ontdvd by divide as not well do as Apicomplexans archaea, fungi), and and bacteria plants (including animals, detail in studied al., of et Weatherall tissues 2000; 2002). al., the et (Tenter in consequence proliferation uncontrolled a rapidly of largely replicate is pathogenesis parasites and individuals, These susceptible 2009). al., et affecting pathogen and 2005) al., et (Snow Ato o orsodne([email protected]) correspondence for *Author Mali. 1805, BP Bamako, Diseases, ikraT Ouologuem T. Dinkorma the of Dynamics ARTICLE RESEARCH ß 3320 2014 May 12 Accepted 2013; December 11 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 2 1 obligate of the thousands such significant pathogens, clinically comprises including 1970), (Levine, parasites protozoan phylum The INTRODUCTION Photoactivation the Complex, after Membrane Inner Schizogony, Endodyogeny, membranes parasites, Apicomplexan WORDS: IMC KEY cell. mother maternal the from of cell, daughters mother of recycling emergence the within by elongation followed IMC daughter during assembly after the of of recovery replication formation the during gondii turnover fluorescence and Toxoplasma the biogenesis monitor IMC imaging, to of photoactivation dynamics mEos2 live-cell and we (FRAP) markers, photobleaching the used IMC form tagged to the recycled fluorescently have or Exploiting process, degraded, IMC? it this is daughter – throughout disappears intact IMC maternal maternal remains the Although mother, membrane cell. the maternal from plasma the emerge of vestiges they behind as leaving daughters membrane assembled plasma their Newly acquire organelles. to daughters rapidly subcellular with elongate new encapsulate which associate (alveolae), filaments but cisternae cytoskeletal membrane as interphase, flattened cytoplasm, during the underlies in IMC membrane develop The (IMC). plasma complex assembled membrane the scaffolding inner are using the endodyogeny), daughters as or known (schizogony multiple mother a which the by within divide in Apicomplexa process phylum the distinctive in protozoa cells, most Unlike ABSTRACT aai eerh&Tann ete eateto pdmooyo Parasitic of Epidemiology of Department Centre, USA. Training 19104, & PA Philadelphia, Research Pennsylvania, Malaria of University Biology, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,32–30doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. niems elbooia ytm hr elcto a been has replication where systems biological cell most Unlike .gondii T. , ahzie.Teesuisrva htthe that reveal studies These tachyzoites. 0 ftepplto olwd (Pappas worldwide population the of 30% oolsagondii Toxoplasma M novstodsic tp – steps distinct two involves IMC oolsagondii Toxoplasma 1,2 aaie epnil o malaria for responsible parasites n ai .Roos S. David and oolsagondii Toxoplasma bqioshuman ubiquitous a – , Plasmodium 1, enovo de * FRAP, , M ignssadtroe during turnover fluorescence and and replication. tachyzoite imaging biogenesis of live-cell dynamics assembly? the monitor IMC used to parasite (FRAP) have as photobleaching after protein recovery we daughter membrane reporter, integral interact during tagged a IMC fluorescently organelles the a does Exploiting other its How is of how regulated? with knowledge and turnover from, our and come and IMC but (Harding 2010; assembly the 2012), does incomplete al., Where al., remains 2014). et Meissner, et function Fung (Beck membrane 2009; identified alveolar al., been et cytoskeletal have Bullen and the IMC proteins Apicomplexan-specific on (Sheffield several membrane and and focused material IMC, have membrane the of maternal studies assembled components plasma Many residual maternal Newly 1968). the any Melton, the from 2008). off emerge up al., ultimately sloughing picking et IMC, cell, the (Nishi mother by schedule delimited strict daughters, according organelles a IMC subcellular rapidly, new to of elongate formation, segregation and the cell cytoplasm coordinating the daughter within of assemble onset complexes and the development parasite At for crucial replication. be to IMC thought al., is organization et Disrupting Stokkermans 2008). 2004; al., 1987). al., et Tremp et and 1996; (Khater shape Chiappino, cell competence integrity, al., invasion pellicle and et alters Morrissette the dramatically 2001; organization Nichols Beckers, give and (Mann that 1997; shape its proteins) and parasite microtubules (intermediate-filament-like subpellicular the alveolins with associated intimately is ebaecmlx(M)(ue l,20a hfil and inner Sheffield 2002a; the al., of et as (Hu mechanism (IMC) known and complex membrane scaffolding biology membrane–cytoskeletal the exploring replication. also parasite are Apicomplexan for parasites These system (endodyogeny). cultivated daughters mother readily two only each assembling within endopolygeny, of 2008). form in al., minimal develop et membranes or (Ferguson and assembly parallel nuclei schizogony membrane daughter termed before when formed is endopolygeny are process nuclei unusual daughter This when 1968). Melton, and eurdfrmtlt n nain(orwlk ta. 1997; al., is et that (Dobrowolski complex invasion motor and actin–myosin Fre motility the for anchors 1969), required leaflet IMC outer Petitprez, The the and ‘pellicle’. parasite of Vivier the immediately called triple 1990; positioned sometimes a is al., of are which et appearance 2008)] the (Foussard giving al., membrane membrane, et plasma the Moore major beneath (Adl 2005; the Alveolata al., – superphylum et alveoli the unifying [cortical feature vesicles morphological Flattened 1968). Melton, ohr(elre l,16;Snu n Cerna and Senaud the 1966; within al., assembled et are (Hepler progeny mother multiple distinctive a which using in replicate mechanism parasites these Rather, fission. binary ´ h M sas ihydnmc n t pta n temporal and spatial its and dynamic, highly also is IMC The eta otepoeso pcmlxnrpiaini a is replication Apicomplexan of process the to Central a ta. 00 Me 2010; al., et nal ne ebaecomplex membrane inner nvitro in ´ ad 01,weestectpamcside cytoplasmic the whereas 2001), nard, making , .gondii T. Toxoplasma ahzie xii a exhibit tachyzoites oolsagondii Toxoplasma ´ 99 Sheffield 1969; , sflmodel useful a

Journal of Cell Science hs tde oue navois uha h M1poen– protein IMC1 daughter the 2008). as al., of such et Nishi alveolins, assembly 2012; on al., the focused et for studies Kono including 2002a; These marker al., cycle, morphological et (Hu valuable cell parasites parasites a the Apicomplexan as IMC of tracking the replication defined the have on studies Previous dynamics replication membrane IMC parasite of during visualization the permits GAP40 RESULTS ARTICLE RESEARCH ou nR tanof engineer endogenous strain to the RH used at in was GAP40–YFP locus 2009) expressing Carruthers, replacements and allelic Huynh for 2009; a membrane responsible al., also complex et IMC (Fre is protein motility which understand glideosome parasite protein the domains, to IMC of transmembrane integral order component predicted an nine GAP40, In employed with IMC. have we inner the dynamics, the of with face associated molecules intermediate-filament-like h rcs fduhe aaieeegnefo h ohr in 1A at Fig. mother, particularly visualize, (cf. the stages to difficult late from be can emergence which IMC1, parasite to daughter contrast throughout of visible process clearly remains the the GAP40 maternal during Third, S1). earlier Fig. 1A parasites Fig. (cf. daughter IMC1 than cycle replicative GAP40 developing Second, merged). with S1, Fig. associates (cf. material ends supplementary basal and 1A; apical the Fig. the of from excluded length is full IMC1 the whereas IMC, labels in GAP40 IMC1 (see First, of ways. localization significant 1 the several Fig. with contrasts in This parasites). illustrated imaging living time-lapse for of as 1 Movie ends, and S1 basal Fig. material supplementary and apical the including A4–F oaie nfrl hogotteprst pellicle, parasite the throughout uniformly localizes GAP40–YFP ´ a ta. 00.TeK8 ytm(Fox system Ku80 The 2010). al., et nal .gondii T. vi upeetr aeilFg S1). Fig. material supplementary ; parasites. i–ii upeetr material supplementary ; aeili erdd eyldo etbhn ntersda body. residual the in behind maternal left as or recycled lost degraded, ultimately is is material which bridge, cytoplasmic by narrow connected remain a Daughters red). for (maturation, mature mother the and from grow daughter to stage, continue final the parasites During 1B,C). parasite (Fig. the SAG1 with antigen surface colocalization by indicated as membrane, plasma agtrprsts(aet)ocr ybdigfo h mother the of from emergence budding the by cell stage, occurs third (magenta) endoplasmic The parasites associated 2008). daughter al., the et (with (Nishi nucleus reticulum) centrosome, and the apicoplast between incorporating Golgi, IMC1). partitioned progressively (e.g. are parasites, cytoskeleton organelles daughter and maternal (GAP40) elongation, membrane growth During aqua) by IMC defined in is the and (shown h, of elongation 3 subsequent of the daughter appearance over basally stage, proceeds the associates second before GAP40 The scaffold above, noted daughter IMC1. As developing below). the the also with to see 2008; adjacent al., nucleus, (Nishi et maternal apparatus Golgi the and (MTOC) center to organizing microtubule apical just occurs blue) as (defined cell assembly daughter of initiation the partitioning). and elongation to apicoplast and Golgi visible prior duplication, centrosome is assembly, (i.e. live-cell IMC time-lapse maternal interphase by corresponding Only determined the during 1). (and The be Movie 2A 1C. material Fig. can Fig. supplementary in in stages form shown cartoon these as in imaging, of and 2, timing and by 1 precise shown Figs as in development, text IMC in colored stages distinct several define to h is tg fICdvlpet ntaino agtrIMC daughter of initiation development, IMC of stage first The signal immunofluorescent GAP40 of pattern the used have We , 8–1 i fe ntain rvdn agtr ihtheir with daughters providing initiation, after min 180–210 ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal t 5 hogotti eot n aee in labeled and report, this throughout 0 0 i ( min 200 ( ( min 45 iue) cl as 5 bars: Scale all figures). throughout maintained the is of cartoons coding color (the maturation and initiation, emergence – elongation, text the in stages defined various the with along membrane (black), plasma parasite (green) the IMC and the of location the relative illustrating Cartoon living (C) in IMC1 cells.) and GAP40 of time-lapse imaging for S1 Fig. material supplementary see be performed; to SAG1 with colocalization enable and resolution provide better samples fixed that (Note maturation. during body residual the from disappears GAP40 maternal that show of arrowheads that IMC1; to prior assembly GAP40 times to corresponding approximately specimens, fixed from images selected gondii the throughout at stages B) various (red, SAG1 plasma protein the membrane and A) alveolin (red, cytoskeletal IMC1 the with B) A and (green, GAP40 protein membrane i.1 tgso h M yl in cycle IMC gondii the of Stages 1. Fig. vii .Arw niaeteiiito of initiation the indicate Arrows ). elctv yl.Sonare Shown cycle. replicative . ooaiaino h integral the of Colocalization ii ,10mn( min 120 ), v ,25mn( min 225 ), , fe emergence after h 2 iii vi ,10mn( min 180 ), m n 6 min 360 and ) t m. 5 0( i ), T. 3321 iv T. ),

Journal of Cell Science nooelk oprmn EC Tmv ta. 03.This the 2013). to or al., endosome, recycling the et to comparable (Tomavo probably is organelle (ELC) compartment endosome-like EERHARTICLE RESEARCH 3322 IMC daughter does GAP40 arrests VP1 especially but 2D, 2005), (Fig. at (BFA) apparatus 2010). insets Golgi al., the al., with et A precisely colocalize et DeRocher not (Agop-Nersesian 2C; brefeldin IMC (Fig. Golgi the development that the with assumed from been long Treatment derived has it be al., 1991), et must al., Morrissette et of 1978; patchwork Torpier a Torpier, 1997; As and IMC. cell the (Dubremetz of parasite vesicles origin the flattened the about for little reveal markers but useful cycle, provide studies Morphological assembled are IMCs Daughter aaie vrtm Fg B e al see 2B; (Fig. time over parasites uigtefnlsae fmtrto.At maturation. cell, mother of the total stages from final emergence The the after during even kinetics. and time, elongation as normal over (defined through initiation linearly of stages with increases earliest highly the daughters from is dividing developing process of this parasites that fluorescence demonstrates in data) raw regular for S1 Table and anandutltebgnigo e elctv cycle. replicative new is a of which beginning plateau, the a until reaches maintained intensity fluorescence emergence), after uniaieaayi fGP0YPfursec rmmultiple from fluorescence GAP40–YFP of analysis Quantitative + vcoa rti )proM2AP 1) protein (vacuolar t 5 n 0mn.GP0i oecoeyascae iha with associated closely more is GAP40 min). 60 and 0 osplmnaymtra i.S2 Fig. material supplementary so enovo de + Rab5 , + uigelongation during fe ntain( h (2 initiation after h 5 rael nw sthe as known organelle t 5 ,fluorescence 0, 5 0), M utb synthesized even the be that kinetics indicating must bleached, normal completely IMC in is with IMC shown maternal elongates the As after parasite and daughter IMC. 3), appears Movie daughter material fluorescence selectively supplementary or (and after maternal 3A Fig. fluorescence the GAP40 either bleaching and replication parasite discussion. further for Robibaro below 2013; see al., 2002); et al., (Jackson et plants in compartment pre-vacuolar loecnercvr Fg B.Cmaigiae rm10and 170 IMC from images daughter Comparing partial 4B). the only (Fig. in of results recovery the elongation fluorescence of portion of process plane length, the a the during later Bleaching within slightly diffuse entire both). to the or able throughout their is membrane, added (or is IMC over IMC daughter entire synthesized rapidly, newly fluorescent recover that that 4A) suggesting uniformly (Fig. IMCs the elongation Daughter during 4. becoming bleached Fig. early in bleached we monitored shown are and assembly, as elongation recovery, during IMC stages fluorescence various of at proteins IMC membrane process daughter of movement the IMC the maternal assess the to throughout of order In portion 3C). a only (Fig. bleaching after only. observed scaffolds was IMC daughter to added newly that is indicating GAP40 IMC, maternal synthesized the in observed was recovery nodrt xmn h yaiso M seby etracked we assembly, IMC of dynamics the examine to order In A4 sual odfuei aueprsts sn recovery no as parasites, mature in diffuse to unable is GAP40 ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal fBA(5 BFA addition of The (C) and (purple). (gray) disappearance parasites IMC interphase as well (red), as maturation and (magenta) emergence (aqua), elongation IMC (blue), indicates initiation coding Color S1). Table Fig. and material S2 supplementary see samples; 24 bti itntfo)teGli(see Golgi the from) to distinct close is initiates (but assembly that reveals GRASP–mRFP (red) marker Golgi the parasites expressing transgenic in (green) of GAP40–YFP for colocalization text Time-lapse see (D) IMC; discussion. the of development arrests further initiation parasite daughter after (right) mean the as 1-h presented of are analysis bins window sliding a of initiation. results parasite The daughter of the time to estimated according aligned images sets time-lapse 14 of from determined dotted respectively), and lines, solid (dashed, grand- parasites and daughter daughter mother, in GAP40 fluorescence of Quantification (B) additional images. for 1 Movie material see supplementary locus); genomic endogenous at the tagged (C-terminally parasites transgenic GAP40–YFP-expressing of imaging lapse GAP40. of dynamics and quantitative imaging Time-lapse 2. Fig. ht iei nagdi h oe ih corner. 5 right bars: lower the Scale in enlarged dashed is the line by white enclosed area The additional images). for 2 Movie material supplementary enovo de m /l t9 i lf)o 8 min 180 or (left) min 90 at g/ml) Fg B.N fluorescence No 3B). (Fig. m m. 6 ..( s.d. A Time- (A) n 5 6–

Journal of Cell Science EERHARTICLE RESEARCH ncnrs otepam ebae aenlGP0ddnot did GAP40 maternal membrane, plasma 1968). the Melton, to and (Sheffield contrast body’ is ‘residual In the material in maternal off unsegregated sloughed emerge, parasites daughter As is but parasites body, maturation daughter residual during into the in incorporated behind and left internalized not is IMC daughters. membrane Maternal developing IMC of of growth unit the remained to per parasites proportionate intensity decreased daughter fluorescence in and but fluorescence time-points constant red min Total (180 5C). developing membrane Fig. the the IMC throughout the daughter to fluorescence disseminated red developing GAP40 2), due photoactivated in Fig. as e.g. increased above, declined, to (see However, material fluorescence the redistribute new of green to addition not 3). (Fig. whereas added does is daughters, GAP40 parasites GAP40 maternal in additional daughter and constant no IMC essentially as maternal remained parasites, and fluorescence maternal red both green elongation, pulse- of a residual process to the analogous During microscopy, experiment. a allowing time-lapse a chase by 5A,B), causes followed (Fig. be Photoactivation red a to fluoresce undergoes fluorescence. it in to red resulting GAP40–mEos2 of to nm, that portion 405 a green at 2009), from al., excited reporter shift et when McKinney change 2010; fluorescent conformational al., et (Baker photoconvertible mEos2 to fused elongation, scaffold. assembly membrane–cytoskeletal of with the concomitant stages time, of early over declines at mobility data its mobile that These highly but is limited. GAP40 brightly is that movement more imply GAP40 remains partial that end and indicating apical fluorescence stained, the apical in but decline recovery, slight fluorescence a shows min 160 aaie eeas rninl rnfce ihGAP40 with transfected transiently also were Parasites rmcnrl(nlahd ohr au ie nFg 7B; fluorescence Fig. the (red), in maturation during lines However, IMC. confirming (aqua the S3), of mothers daughters Fig. of material (unbleached) that supplementary to control comparable rate, from linear approximately an elongation, throughout at increased mothers bleached from parasites min) 488 (cf. progressively maturation daughter became during 3), mothers dimmer Fig. bleached min; maternal from 255 of emerging (cf. parasites independent elongation was during parasites bleaching IMC daughter whereas of Moreover, images 4). min intensity Movie 383 the material (cf. through supplementary mothers 7A; bleached Fig. the progressed in mothers of (unbleached) accumulated daughters control in cells briefly from not but daughters GAP40 supplementary of daughter of cytoplasm the 7; pool As in (Fig. cytoplasmic a 4). parasites maturation, daughter Movie after GAP40 of material of emergence dividing accumulation in cytoplasmic IMC the maternal monitored the bleached in and we shown cells 6B, as Fig. GAP40, in maternal cartoon from the derived actually was 6C. 2001) GAP40 Fig. al., al., in et shown (Robibaro et as 2013), ELC (Rabenau al., the et VP1 proM2AP in Tomavo 2001; accumulates with and GAP40 Colocalization 2010) that indicated min). al., 0–60 of et stages was 2D, early (Miranda (Fig. the apparatus during GAP40 Golgi assembly noted the previously IMC as – of min), from 300 distinct accumulation 2D, clearly (Fig. but cytoplasmic – with associated This min)]. column 288 6A, [Fig. 1A Fig. cells and emerging of daughter cytoplasm the maturing coincided in GAP40 body of residual accumulation the an with from IMC maternal of disappearance eani h eiulbd Fg C i.6,column 6A, Fig. 1C; (Fig. body residual the in remain uniiaincnimdta h loecneo daughter of fluorescence the that confirmed Quantification of pool cytoplasmic late-appearing this whether determine To ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal vi ,B vi i.2 upeetr aeilMve1(213– 1 Movie material supplementary 2; Fig. ; aueprsts cl as 5 bars: in Scale diffuse parasites. to mature unable is GAP40 suggests that also line) yellow by dashed enclosed the area (the the IMC of maternal part only Photobleaching (C) images. for additional 3 Movie material See supplementary (B). IMC to maternal added the not is GAP40 synthesized is IMC synthesized daughter the that elongation, indicating or initiation on IMC impact daughter no has line) IMC (yellow maternal the of photobleaching is IMC assembled daughter The 3. Fig. enovo de enovo de vi enovo de lovsbein visible also ; . n htnewly that and A Laser (A) synthesis vii .The ). 3323 m m.

Journal of Cell Science EERHARTICLE RESEARCH 3324 ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal hne nyatrpoociain.Saebars: Scale 5 photoactivation). red after (shows only fluorescence channel red illustrating of Cartoon diffusion (C) the IMC. the within diffusion suggesting (arrowheads), elongation even uniform during fluorescent remains red fluorescence a Red to protein. reporter a this converts of (A,B) portion initiation after at times light various violet to GAP40–mEos2 expressed IMC. the elongating within movement protein GAP40–mEos2 indicates of Photoactivation 5. Fig. m m. rohas,oigt combination of a to owing (closed arrowheads), elongation of stages early at rapidly recovers parasites daughter elongation. daughter during declines redistribution GAP40 4. Fig. elw cl as 5 bars: Scale in yellow. outlined are areas Bleached arrowheads). open (B, post-initiation after applied is when bleaching redistribute fully to is unable fluorescence but (A), trafficking enovo de xoueo transiently of Exposure ytei n ifso or diffusion and synthesis A4 loecnein fluorescence GAP40 m m. , 5 min 150

Journal of Cell Science lnaigduhesbcm nraigygendet the to while due 5), green (Fig. during increasingly previously IMC became noted maternal daughters As of elongating 8A,B). recycling (Fig. apparent maturation the investigate 3). Movie material supplementary normal right; quantitatively with 7B, results replicating were (Fig. parasites the kinetics of parasites and those replicating S3) from indistinguishable slowly Fig. 7B, 4; from (Fig. more-slowly recycling Movie obtained and material the maturation supplementary atypical, for elongation, left; resolution IMC time Although of were increased analysis phototoxicity provided analysis). severe parasites more replicating from to owing divide excluded (parasites to photobleaching ceased laser conditions that environmental during phototoxicity to in specimen mild differences from and/or subtle variability to some observed due we specimen, but Nishi 2008), 1995; al., al., et et Fichera 2010; al., et (Behnke previously reported EERHARTICLE RESEARCH A4 sitraie uigmtrto n eyldinto recycled and 7C. Fig. maturation in illustrated during as IMCs internalized daughter maternal that is to hypothesis the due GAP40 support body addition strongly residual GAP40 observations the of These from cessation IMC the maternal The and of 7B). disappearance Fig. precisely the coincided in material with this lines of yellow disappearance and versus appearance (gray mothers unbleached mothers bleached (solid from rise symbols). cells (open to daughter slowed of continued that controls whereas unbleached symbols), from daughters of h A4–Es uinpoenwsas xlie to exploited also was protein fusion GAP40–mEos2 The h elctv yl fR strain RH of cycle replicative The from emerging daughters in only arose fluorescence Internal .gondii T. stypically is enovo de synthesis. , 7h,as a iil ntersda oya h ieo emergence of min), min). time (270 (210 IMCs region the daughter ELC at and the Golgi in body the appeared through residual IMC later trafficking maternal but the The min), in (180 8C). (Fig. the during visible the in IMC followed lost daughter was be When could labeled fortuitously IMC time. of maternal was absence the over of GAP40 fate plasmid the as fluorescent interphase, studies, transfected of expression the synthesis transient lost from parasites (arrows). came parasites IMC daughter daughter (red, min). (390 the IMC of mature throughout parasite incorporated daughter appearance the became mature GAP40 the in maternal with Recycled daughter and parallel GAP40 maturing arrowheads) in the maternal) arrowheads), into (open GAP40 (solid cytoplasm the of body Over internalization lost gradually arrowheads). residual transient was (solid IMC the developing maternal body min), from 260 the residual (cf. the h off into 1–2 sloughed of subsequent was incorporated form – (red) the not IMC in maternal the was as including lost, – that was daughters morphology but material green maternal clearly stage, were this the min At 200 (arrows). at red beginning cell not parasites mother the Daughter from images). background emerge min near to 170 to in declined red (arrows had the growing levels but IMCs parasites, daughter of daughter the of By in fluorescence min). amount evident 135 was throughout 8A, fixed fluorescence Fig. green a (cf. dispersed IMC because daughter GAP40 discern to photoactivated became fluorescence difficult red material, increasingly synthesized newly of addition ute upr o h eyln fmtra M into IMC maternal of recycling the for support Further ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal hrl fe mrec rmthe from emergence after parasites shortly daughter of region Golgi ELC the and in GAP40 of appearance ( both elongation during ELC IMC the the through to traffics (green) IMC. GAP40 daughter (C) the to maternal body the residual from IMC of recycling suggest parasites emerging in arrows (red); membrane the plasma and parasite (green) IMC of relative location the illustrating Cartoon (B) ( min 225 ( eiulbd.Saebr:5 bars: Scale body. residual ( mother auain(avg ftematernal IMC; the of (salvage maturation times to corresponding approximately are shown, specimens fixed from images gondii the throughout stages at various (red) SAG1 protein membrane plasma and protein (green) IMC GAP40 the of Colocalization (A) maturation. and emergence parasite daughter the during in ELC appears the and from body lost residual is GAP40 6. Fig. iii ,10mn( min 190 ), vi elctv yl.Selected cycle. replicative .Arw niaethe indicate Arrows ). vi t 5 vi ;arwed niaethe indicate arrowheads ); n 6 i ( min 360 and ) 0( enovo de , iv i ,3 i ( min 30 ), post-activation, h 2 ,20mn( min 200 ), synthesis; ii ,10min 150 ), vii v ), ). m T. m. ii 3325 and )

Journal of Cell Science EERHARTICLE RESEARCH 02 erlye l,21;H ta. 02;H ta. 06,but 2006), al., 3326 et Hu 2002a; al., et Hu IMC 2012; al., the et al., Dearnley on et 2012; Anderson-White previous focused 2011; al., Most yet have et (Anderson-White replication (as homeostasis. cytoskeleton Apicomplexan play and/or also on storage might alveolae studies (the in the IMC roles with the motility , analogy undefined) al., and By et parasite 2004). (Gaskins Soldati, of invasion molecular with and and anchor Keeley associated attachment to 2004; cell serves are host also glideosome), IMC (Tremp that mature 1968) Melton, the complexes and 2008), Sheffield al., 2004; et al., et parasite (Khater of maintenance shape the and a assembly providing cell to daughter membrane– addition for In scaffold a IMC. process, using the as mother unusual known structure the cytoskeletal highly within a cells daughter by assembling replicate parasites Apicomplexan DISCUSSION 5 bar: Scale maturation. showing during Cartoon witho (C) IMC grow bleaching. maternal to before but the continue parasites (gray) parental of mothers controls of stage salvage unbleached bleached fluorescence elongation from and from the maturing the elongation indicate those (magenta) daughters diamonds through during whereas in open emergence mothers bright, and observed (aqua), unbleached Filled remain is elongation (yellow). versus fluorescence controls (blue), mothers bleached cytoplasmic unbleached ma initiation Transient from from supplementary fluorescence. IMC emerging also maturing additional indicates daughters see daughters accumulating coding in (left; versu as Color 7A comparable symbols) emergence, 3). Fig. is (solid after Movie in fluorescence parasites diverges images material that control to supplementary Note unbleached corresponding the (right; arrowhe (red). from experiments, from (filled 3A maturation daughters independent parent those Fig. on in control than and based fluorescence unbleached with brighter 4) are IMC the elongating are Movie Panels of from IMCs mother symbols). daughters Quantification daughter (open unbleached in (B) the parasites the evident arrowhead). bleached of from is (open fluorescence maturing fluorescence parent comparable cytoplasmic daughters bleached reveals phase, min), the maturation imaging (308 the Time-lapse emergence During control). after mother. internal but, bleached an mothers (providing unbleached emergence. not versus their was bleached after sister daughters developing its into of recycled that is IMC Maternal 7. Fig. h nerlICmmrn rti A4 (Fre GAP40 protein that membrane IMC vesicles integral 1991).the flattened al., et of (Torpier patchwork assembled is the membrane how IMC the of comprises little know we lnaininvolves the elongation that supplementary revealed 8), 7; 5, of (Figs IMC 4, photoactivation and 3, S3) (Figs Fig. material in photobleaching microscopy, video with material time-lapse supplementary combination by 6; analysis 2, Careful S1–S4). 1, Figs (Figs elongation, maturation initiation, – and stages emergence developmental distinct four define rcse ngetdti Fg B i.7;splmnaymaterial supplementary 7B; analysis Fig. these 2B; and of (Fig. timing the detail imaging the great track in modern quantitatively of processes to Using possible recycling is 8). it 7, techniques, and (Figs salvage IMC maternal involves maturation emergence m nodrt ietyeaieICmmrn seby eused we assembly, membrane IMC examine directly to order In m. A h nieICo n aaiewspoolahd(elw,whereas (yellow), photobleached was parasite one of IMC entire The (A) ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal .gondii T. ahzie sasmldi w itntpae – phases distinct two in assembled is tachyzoites enovo de ytei Fg ) hra post- whereas 3), (Fig. synthesis enovo de ´ a ta. 00 to 2010) al., et nal ytei fteIMC the of synthesis o bleached not d u not but ad) in and s but terial ut

Journal of Cell Science eosrtsta ntainadeogto cu lotentirely almost occur elongation through and initiation that demonstrates IMC. maternal the EERHARTICLE RESEARCH eot httemtra M ndividing in from IMC daughters fluorescent maternal of the previous brightly that fluorescence supports observation reports the This become 7B). to (Fig. mothers comparable parasites unbleached – daughter 3A) (Fig. recover, to emergence cycle, and developmental elongation ( IMC initiation, Behnke entire parasite 2008; the daughter al., highlights including et but Nishi 2010), with 1995; al., consistent al., et remarkably et (Fichera is studies (open S4) previous region Fig. ELC development and tachyzoite material Golgi for timecourse (supplementary the resulting through The IM recycling 5 S3). maternal min), bars: S2, the 180–210 Scale Figs only arrowheads; (arrows). bod where (closed IMC residual Parasites body daughter the (C) residual the in photoactivation). th the off of after throughout in sloughed labeling only disperses IMC is and channel parasites display IMC min) red text) daughter Maternal 210 min) (shows in (see min). arrowheads; 260 fluorescence IMC loss (170 arrowheads; red (open red plasmid GAP40 system of of to (green) endomembrane diffusion amount due daughter synthesized the the small newly into illustrating The by enters Cartoon it min). diluted which (B) (35 rapidly from red min), is to 200 and arrowheads; green min) (closed from maturation. (135 GAP40 during IMC of daughters daughter into portion developing GAP40 a maternal converts of (lightning) recycling formation confirms Photoactivation 8. Fig. iprewti h agtrICdrn h first to the able is during GAP40 IMC that daughter reveals also the in FRAP expansion within 2002b). by disperse grows al., microtubules subpellicular IMC et assembly of the assembly (Hu of that processive the suggesting process to 8), contrast the 4, and throughout 3, (Figs (Vivier uniformly IMC IMC daughter maternal discussion. Early the further for might 1968). of below see IMC Melton, outgrowth but 1969), daughter and Petitprez, an the Sheffield as that al., 2002a; originate et suggested (Dubey al., studies cell et ultrastructural mother the Hu from 1998; daughters of to assembly emergence daughter of the stages earliest very the from intact remains , lahn ftemtra M ro oduhe assembly daughter to prior IMC maternal the of Bleaching ti neetn htGP0apast eaddt h entire the to added be to appears GAP40 that interesting is It . ) olwdb auain( maturation by followed h), 3.5 enovo de ytei.Atog lahdmtra M fails IMC maternal bleached Although synthesis. , ) nldn eyln of recycling including h), 2 .gondii T. tachyzoites , hof2h m m. otne oepn fe mrec,truhslaeo the of salvage daughter through that demonstrates emergence, after clearly Photobleaching after and IMC. also maternal expand during membrane IMC into to form daughter that the continues that to incorporated and report now continue development, is We emergence. micronemes during mitochondrion and late rhoptries the very of daughters residue that developing heme revealed polymerized have the on In studies includes Previous (hemozoin). 6). digestion hemoglobin body (Fig. material body residual maternal residual the remaining maternal the any the in leaves in behind IMCs and daughter membrane developing plasma clothes that 2002). process al., of et assembly Mann 2007; and al., et the rafts (Johnson with lipid complex glideosome association cytoskeleton, the of IMC consequence and a microtubule as declines mobility presumably GAP40 (data time, that cell clear with host is the it on Nevertheless, to shown). effects the toxic not indirect are to between as due inhibitors small parasites synthesis of distinguish intracellular trafficking, protein analysis and the to vesicle-based spots precludes photobleached parasites or failed intracellular of continuously motor- have motility al., et is versus Mann studies 2002a; diffusion which upon contrasts al., Further et This IMC1, fixed (Hu 2002). assembly 3C). protein essentially (Fig. IMC during cytoskeletal parasites remodeled is the mature and in with immobile 4B) and (Fig. increasingly emergence becomes elongation but 5), during Fig. 4A; (Fig. assembly e aaie liaeyeeg ybdigoto h ohr a mother, the of out budding by emerge ultimately parasites New ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal A ciaigGP0mo2a h ne fdaughter of onset the at GAP40–mEos2 Activating (A) nroute en oteduhe M (arrows). IMC daughter the to Plasmodium Toxoplasma svisible is C parasites, 3327 e y

Journal of Cell Science aenlICfo h eiulbd slkl opoedby proceed to of likely removal is ER the body IMCs, the residual daughter with the the fusion from to 2013) the GAP40 IMC ER–Golgi– body trafficking al., maternal the observed and residual by et were the vesicles proceeds treatment, (Tomavo from no IMC As ELC BFA the pathway. to the ELC by trafficking with that The right) GAP40 argues min). 2C, of IMC 390 (Fig. association 8A,C, salvage (Fig. maternal 8C, incorporated Fig. IMC IMCs both becomes of and however, min; daughter 200 disruption body both residual of maturation, 8A, throughout (Fig. point the the from During cells at disappears body daughter residual min). 180 the of in IMC emergence maternal all leaving ohr u rudfra al aenlcnrbto.O the IMC the On daughter contribution. the of that maternal that demonstrate we early from imaging, an live-cell derive of for basis might argued but IMCs mother, daughter that suggested ‘maturation’. cycle developmental appreciated newly parasite this the termed of have We phase 7). (Fig. of salvage IMC but maternal to mother, attributable the entirely the almost of is independent emergence after is growth emergence to prior growth IMC ARTICLE RESEARCH viaiiyo odmresfrbt h yokltland cytoskeletal degree high the 3328 the The and both strategies. 1), (Fig. for therapeutic IMC the new markers of yield components good membrane also of might availability IMC understanding the better proliferation, rapid of of consequence pathogenesis direct parasite a Apicomplexan is that fact the 1966). the organization and al., structural motility, parasite within and et in IMC Hepler the of parasites 2000; importance the al., Given ring-stage et (Bannister erythrocyte into infected as differentiate observed degradation IMC merozoites rapid maternal the in the into of maturation of insights degradation during rapid replication The IMC equivalents. intraerythrocytic IMC the new 15 during the typical falciparum diminishes daughters is 16 certainly of production (as the daughters as recycling, multiple but from savings unknown, potential of is parasites production during these recycled of the is maturation IMC the and the emergence to Whether the prior 2008). by al., divide et many nuclei daughters (Ferguson which IMC (and in multiple schizogony, stages produce or endopolygeny life-cycle parasites) Apicomplexan Other other efficient. be highly recycling will endocytosis). it and trafficking secretion IMC membrane dense-granule other defined, and to (including maternal processes recycling, pathways are these and in of biogenesis relationship IMC markers protein the explore further this additional to interesting late of As the involvement during recycling. suggesting 2013), IMC possible al., et the (Jackson the (Stx6, assembly to cell Golgi) 6 daughter trafficking the of to stages syntaxin vesicular Agop- ELC of disrupts the 2010; from also Overexpression al., transport retrograde 2009). et regulates which al., (Agop-Nersesian et unclear organellar Nersesian is between relationships trafficking, effects and causal the vesicular these complex but perturb cytokinesis, glideosome adequate and the and of segregation Rab11B of lack biogenesis, assembly and the IMC the to Rab11A disrupt due Dominant-negative proteins part markers. in molecular recycling, and formation 5). Movie material supplementary 2D; Fig. replicative occurs synthesis parasite IMC the early all during above, late that noted but only As components, cycle. occurs maternal recycling salvaged from this derived indeed is trmisucranwa ahnr sivle nIMC in involved is machinery what uncertain remains It 1969) Petitprez, and (Vivier studies ultrastructural Previous Toxoplasma o xml)ncsiae h rdcino tleast at of production the necessitates example) for , ahzie iieb noygn,making endodyogeny, by divide tachyzoites enovo de nroute en Toxoplasma M seby(i.2,lf)and left) 2C, (Fig. assembly IMC oteGliadEC(i.6C, (Fig. ELC and Golgi the to ih rvd useful provide might Plasmodium enovo de P. , ieto n eunig fe ierzto with linearization BsiW After sequencing. and digestion into nvriyo ihgn(un n artes 09] Plasmid 2009)]. Carruthers, and (Huynh Michigan of University pfamn rmte3 the from fragment bp pLic ilpr,Bleia A tafnlcnetaino 0.5 of concentration final a at MA) Billerica, Millipore, Fg B upeetr aeilFg 2 3 rvdsuseful provides S3) S2, screening. small-molecule Figs for material tools defined be supplementary can cycle 2B; cell parasite (Fig. the which with resolution time of upeetr aeilTbeS,aditgainit the into integration and S2, in Table 1358-bp-spanning sequences material of amplification supplementary PCR ( plasmid by replacement engineered allelic The Plasmids 37 at cultivated were (HFFs) fibroblasts foreskin Human parasites and Cells METHODS AND MATERIALS F el eegont ofuneo 2m ls oesis infected coverslips, glass 22-mm on confluence with to grown were cells HFF microscopy Immunofluorescence eu NS hroSinii,Wlhm A,5 /lpenicillin, U/ml 50 MA), Waltham, to Scientific, NY) calf 50 Thermo newborn Island, 10% (NBS, Grand with supplemented serum Technologies, Technologies), Modified (Life Life Dulbecco’s 199 glucose Medium (DMEM, high of mixture Medium 5:1 a Eagle’s in 1995), al., et (Roos qipdwt PaSp 100 UPlanSApo a with equipped AL). mounted Birmingham, and Biotech, (Southern PBS Fluoromount-G with in once slides and glass X-100 on Triton 0.25% with twice washed for incubated then 4 were with samples min labeling, 10 DNA for For stained Technologies). (1:5000; and Life antibody PBS anti-mouse-IgG coverslips of goat in Alexa-Fluor-594-conjugated 2002)] X-100 in University h Triton al., 0.25% 1 Ward, et with times Wichroski Gary three 2001; washed by were Beckers, solution; provided and blocking (Mann kindly Vermont in [1:2000, 1993)] al., [1:400 anti-IMC1 et serum (Mineo anti-SAG1 or College bovine Dartmouth Kasper, monoclonal 3% Lloyd for by in provided murine incubation kindly temperature After with X-100. room Triton h at 0.25% 1 plus h V 1 X-100 fraction for Triton (BSA) (0.25% albumin blocked min and 0.05% 15 PBS) and for formaldehyde in permeabilized (4% PBS), min in 15–20 for glutaraldehyde fixed then were Coverslips uiiidamshr otiig5 CO 5% containing atmosphere humidified ehoois,1 etiatvtdftlbvn eu FS Thermo (FBS, above). (Life Medium serum (as Glutamax bovine antibiotics Essential mM and fetal 2 Minimal Scientific) heat-inactivated with with 1% supplemented replaced Technologies), Technologies) was Life with medium (MEM, cells growth the of this inoculation to prior Immediately ih50 with gondii T. ihmo2apiida an as in amplified YFP-HA mEos2 the with replacing by ( engineered GAP40 with 50 with Esvnui knl rvddb ihe aisn lrd State with Florida transfected were Davidson, Parasites 50 2010)]. Michael al., et by (Kanchanawong University provided [kindly mEos-vinculin and dilution in limiting sequences by ACP isolated expression. transgene were for microscopy plaques fluorescence Clonal by screened 1995). al., et tnad(o-piie)cdn sequences. coding (non-optimized) standard , Tg 8hps-rnfcin l rngns(F,mhry Es)utilized mEos2) mCherry, (YFP, transgenes All post-transfection. h 18 mgn a efre na lmu X0ivre microscope inverted IX70 Olympus an on performed was Imaging Plasmid m m E9295;TxD) sn h rmr hw in shown primers the using ToxoDB), ME49_249850; IMC1mCherry- /lsrpoyi n 25 and streptomycin g/ml fpamda bv n rnin rnfcat eeeaie at examined were transfectants transient and above as plasmid of g p frIC) 15 IMC1), (for I .gondii T. Cer.i.HR l lsiswr ofre yrestriction by confirmed were plasmids All mCherry.Lic.DHFR. m m ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal fpamdadwr eetdi 25 in selected were and plasmid of g /lxnhn GP0 r1 or (GAP40) xanthine g/ml ahzie HyhadCruhr,20)wr electroporated were 2009) Carruthers, and (Huynh tachyzoites ptub 9 6daiio2peyidl iyrclrd DP,EMD (DAPI, dihydrochloride ,6-diamidino-2-phenylindole p ahzie n nuae t37 at incubated and tachyzoites GAP40YFPHA- Bgl F.i.X knl rvddb enCarruthers, Vern by provided [kindly YFP.Lic.HXG II– dhfr ptub 6 Avr 10 HRwsegnee iial,uiga1950- a using similarly, engineered was DHFR 9 ACP-YFP-HA/ 6 I.Plasmid II). n of end rsl avse RH harvested freshly Avr sag m Tg /lgnaii Lf Technologies). (Life gentamicin g/ml II– 6 A a nierdb elcn the replacing by engineered was CAT M1( IMC1 pLic Afl i-meso betv (NA1.4), objective oil-immersion m ptub sag yiehmn IC)(Roos (IMC1) pyrimethamine M Ifamn rmteconstruct the from fragment II GAP40YFP- CAT Tg ptub GAP40mEos2- 2 speiul described previously as , E9214)integrated ME49_231640) m ˚ sag D o ute 82 h. 18–24 further a for C GAP40YFPHA- /lmcpeoi acid mycophenolic g/ml Ku80 .gondii T. Kas Nsie l,2008) al., et (Nishi dhfr D frGP0 or GAP40) (for I XPTstrain HXGPRT XPTwas HXGPRT m sag g/ tachyzoites, ˚ ne a under C m A was CAT Tg linPBS, sag GAP40 CAT Lic

Journal of Cell Science Mguaie 0 /lpncli,100 penicillin, U/ml pyruvate, 100 sodium glutamine, mM mM 1 2 FBS, 1% with supplemented Technologies), of (Life (MOI) infection of multiplicity rcso,Isqa,W)addcnovd(is1 ny sn the out-of-focus of using 0.2 or effects 3 only) 0.1 the was 6 size minimize Step 1, (Applied to fluorescence. (Figs software algorithm, deconvolved Image iterative SoftWorx and nm. constrained 632/60 DeltaVision WA) and 520/ Issaquah, using nm and 572/35 Precision, nm captured 360/ were 470/40 RFP were were were or GFP mCherry DAPI stacks for for for and respectively; nm, used nm, 40 filters 455/50 cooled-CCD and emission monochrome nm HQ and 40 CoolSNAP excitation a The and lamp camera. arc xenon W 300 ARTICLE RESEARCH a are u sn iisfwr sdsrbdaoe xetthat except above, IMC. daughter and described maternal the to as addition in and software parasites, emerging daughter for maturing Fiji collected was intensity using fluorescence GAP40 cytoplasmic out presented) time-series carried individual the was Quantification (from Devices). fluorescence (Molecular GAP40–YFP stacks 7 of Image MetaMorph nm). using 435 processed wavelength dye, were nitrogen- 440 a (Coumarin laser of consisting dye CT) pumped ablation Windsor, South Galvo Technology, MicroPoint (Andor a system using above, spinning-disk described IX-71 microscope Olympus confocal same the on performed was Photobleaching photobleaching after recovery Fluorescence 37 at h 12–16 for incubated and gentamicin], ml with infected WI), Verona, glass-bottomed (Ibidi, 35-mm in cultivated dishes were monolayers cell HFF Confluent imported microscopy Time-lapse were and 2012) preparation. figure al., for et PowerPoint (Schindelin into software Fiji open-source aaa htwseupe ihadgtltmeaue CO temperature, ON, digital TC a Chamlide Guelph, with a Instruments, equipped to was Cell transferred that (Live Canada) then were chamber Samples environmental 7. stage-top pH and HEPES phototoxicity) laser mM minimize 25 (to FBS remove 10% (as with (to DMEM supplemented Phenol-Red-free cations above) fresh divalent in lacking incubated and PBS parasites) warm extracellular with rinsed were cultures oto ntadequilibrated and unit control betv N14,CU1 cne Ykgw,Nwa,G) and GA), 5 planes Newnan, NJ). (26 Bridgewater, stacks (Yokogawa, (Hamamatsu, camera scanner EMCCD 100 C9100-13 CSU-10 UPlanSApo (NA1.4), a with objective equipped microscope confocal acltdb ldn-idwaayi,poigsmlswti hourly per within was (2 samples fluorescence pooling parasites IMC initiation (e.g. analysis, daughter vacuole). windows sliding-window of per by formation (4 shortly calculated grand-daughters the parasites and to ‘mother’ vacuole) through interphase invasion from of after stages analyzed, all representing were vacuoles Multiple replication etc. min 600–615 at initiation ( to estimated reliably be at can emergence values parasite Offset daughter S2A). Fig. , and material supplementary S1 in column Table (‘Offset’ initiation parasite daughter of quantification). Table for material S1b (supplementary and images structures for IMC S1a daughter resolvable representing lines maternal, distinctly drawn manually all all total on and based for length IMCs, perimeter subtraction) grand-daughter and/or collect (for background to time-point used (after was each from fluorescence software selected At Fiji was fields. and plane stack eight image h the central in 9 the continuous vacuoles vacuole) a 15 parasitophorous over each of collected each were for above) as session planes, (26 stacks image presentation. figure Fiji for and enhanced MetaMorph contrast were using images emission processed (Spectral Some the software. and RFP using PA), or (Molecular Downingtown, min, mCherry 7.7.4 30 MetaMorph Devices, for using collected or ET630/75 was 15 Data and Research). every Applied GFP power) for laser ET525/50 filters (1% nm 561 and n m 5 of timing coincident closely the by confirmed as resolution, min 15 ielpeiaigwspromdo nOypsI-1spinning-disk IX-71 Olympus an on performed was imaging Time-lapse niiultm-as eiswr lge ae nteetmtdtime estimated the on based aligned were series time-lapse Individual time-lapse 9–12 2), (Fig. development IMC of quantification For ,stsyn yus apig mgswr ute nlzdusing analyzed further were Images sampling. Nyquist satisfying m, –4 e upeetr aeilTbeS o apesizes). sample for S1 Table material supplementary see 6–24; 6 0.2 m t tp)wr curdb xiaina 8 nm 488 at excitation by acquired were steps) m 5 0 6 , 0mn,adi rsne stemean the as presented is and min), 30 eoedt acquisition. data before h 2 , , 9–1 i,gadduhe IMC grand-daughter min, 195–210 : i MMlcigPeo Red Phenol lacking DMEM [in 2:1 m n custo et was depth acquisition and m m /lsrpoyi n 50 and streptomycin g/ml .gondii T. ˚ .Pirt imaging, to Prior C. 6 ahzie ta at tachyzoites 2 oil-immersion n humidity and m image m 6 , s.d. m 2– g/ o 21 t37 at h incubated 12–16 and above, for described as dishes glass-bottomed on grown cultures olg,Hnvr H n ayWr Uiest fVrot ulntn T for VT) Burlington, Vermont, of (University Ward (Dartmouth Gary Kasper and Michael Lloyd NH) MI), Hanover, FL), Arbor, Tallahassee, College, Ann University, the Michigan, State for of (Florida CA) (University Davidson Francisco, Carruthers San Vern (Genentech, clone; Date GAP40 Shailesh Laboratory, and Chemical India) (National Maharashtra, Shanmugam Dhanasekaran thank We Acknowledgements with studies, transfected photoactivation For Photoactivation anse,L . okn,J . olr .E,Kiha .adMthl,G H. G. Mitchell, and S. Krishna, M. E., R. M. Fowler, M., Falk, J. Hopkins, and H., L. 3rd Bannister, W., R. Buckheit, M., S. and Baker, J. P. Bradley, M., Meissner, C.-T., Chen, R., J. Beck, A., B., Lorestani, Anderson-White, T., Szatanek, K., Cheng, D., F. Ivey, R., B. Anderson-White, P. J. D. Ferguson, J., B. Foth, G., Langsley, S., Egarter, C., Agop-Nersesian, Kretzschmar, M., Rauch, F., Rached, Ben B., Naissant, C., Agop-Nersesian, R., O. Anderson, A., R. Andersen, A., M. Farmer, B., G. A. Simpson, M., S. Adl, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.147736/-/DC1 online available material Supplementary material release. Supplementary immediate for PMC [grant in Heath Dissertation Deposited of SAS D.T.O.). Institutes an (to National and Fellowship US D.S.R.]; Completion the to by R01-AI49301 part, R37-AI28724, in numbers funded, was work This preparation. Funding manuscript and all analysis to data equally design, contributed experimental authors of both D.T.O.; aspects by conducted were experiments All contributions Author interests. competing no declare authors The interests Competing design. experimental on advice for IN) Murray John Bloomington, and University, PA) PA), (Indiana Philadelphia, Philadelphia, Pennsylvania, of Pennsylvania, (University and of Lampson Materials (University Michael under Gallagher specified Kim as and reagents Methods; antibody and plasmids sharing aulgscnrle ntadwsequilibrated was and unit a as well controller as unit gas control humidity and was manual temperature that digital CT) a Hamden, with equipped Instruments, (Warner chamber environmental top uniaiemaueet eepromduigupoesddata. unprocessed using for performed nm were all measurements using quantitative 617/73 but presentation figure and for GFP enhanced only contrast for were images nm time-points Some TRITC). 525/50 selected filters emission nm power; at 561 laser and (5–25% 488 stacks) at collected was Data (no Devices). (Molecular 7.7 MetaMorph acquired were crystal images diode-pumped nm). mW 405 of 50 wavelength a DL405-050-O, (Roper model employs system (CrystaLaser iLas2 that laser the France) and Paris, Technology) Scientific, (Andor camera EMCCD 100 IX-81 iXon3 897 UPlanSApo and (Yokogawa) Olympus scanner an CSU-10 (NA1.4), with an objective oil-immersion equipped using microscope performed confocal spinning-disk was Photoactivation acquisition. 20) re lutae ud oteutatutr fPamdu falciparum Plasmodium of ultrastructure the to stages. guide blood asexual illustrated brief on A dynamics (2000). protein and microscopes. arc-based cell mercury tracking wide-field standard proteins: fluorescent photoconvertible division. M.-J. J. the Gubbels, M. into Gubbels, assembled and sequentially gondii. N. is Toxoplasma Sahoo, proteins of filament-like cytoskeleton J., intermediate D. of Ferguson, family J., Rab11B. C. GTPase Beckers, specific the by Pathog. transport PLoS vesicular on M. dependent Meissner, cytokinesis. and apicomplexan of completion e1000270. Langsley, for and M. Meissner, required P., J. D. Ferguson, G. R., Menard, S., S. Thiberge, A., Fredericq, A., R. protists. Fensome, of G., Brugerolle, the S., S. al. Bowser, et R., J. Barta, wn otesotnospoolahn rpriso Es,single mEos2, of properties photobleaching spontaneous the to Owing 20) a1Acnrle sebyo h ne ebaecmlxis complex membrane inner the of assembly Rab11A-controlled (2009). 20) h e ihrlvlcasfcto fekroe ihepai on emphasis with of classification level higher new The (2005). ora fCl cec 21)17 3033 doi:10.1242/jcs.147736 3320–3330 127, (2014) Science Cell of Journal n.Rv elMl Biol. Mol. Cell Rev. 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