Journal of Cell Science lglu,wt t itledatce oteodflagellum progresses, old cycle the cell to As existing (FC). attached the connector end to flagella distal posterior the assembled its through cell is the with flagellum During flagellum, 2003). new al., a kinetoplast the et cycle, to (Ogbadoyi attached seeded physically DNA) flagellum are (mitochondrial that single bodies a basal contains the cell by parasite each flies, tsetse Sharma the 2011; to al., coupled et (Rotureau 2008). tightly length al., immotile is body et length cell intracellular, and its length during and the FAZ small varies development a in also cycle length only life present Flagellum and cells. is parasite amastigote is motile flagellum flagellum extracellular, long residual a the that in so In present regulation 2010). for tight al., important et undergoes is (Gluenz flagellum length style flagellum life the parasitic of their trypanosomatid control in the interactions flagellum and the parasites for size al., observed organelles, et host-parasite been eukaryotic Oberholzer single many has cell Like regulation 2007; 2006). al., and al., a motility, et et Ralston Engstler cell 2011; of 2006; evasion al., normal presence et for (Broadhead immune the important single-cellular is division, by These which characterized 2007). flagellum, al., are et health organisms (Mathers global to threat productivity a remain and still that pathogens veterinary and cruzi Trypanosoma ieolsi aaie including parasites Kinetoplastid Introduction FAZ and flagellum body. coordinating cell joining the By zipper, to elongation. molecular flagellum FAZ a the from like to transmitted acts words: biogenesis is Key membrane flagellum information flagellum morphology the linking cycle, procyclic on and cell in a FLA1BP the membrane identified (FLA1), and during was cell 1 membrane assembly (FLA1BP) protein cell to protein the adhesion membrane FLA1-binding the on flagellum novel flagellum FLA1-truncation how FLA1 the a a understand between of of flagellum, To immunoprecipitation interaction organization the By morphogenesis. The division. domain to cell cell and the mislocalized and and cycle division that attachment examined cell flagellum cell mutant we for the positioning, essential coordinated, during organelle protein FAZ, are transmembrane in and glycosylated, assembly role flagellum and pivotal FAZ of proteins a and assembly adhesion plays membrane coordinated flagellum by development, The cortex cycle complex. cell (FAZ) life the to zone the attached attachment is flagellum that intracellular flagellum membrane-bounded an single, a have trypanosomes African Summary 10.1242/jcs.113621 doi: 520–531 126, ß Science Cell of Singapore Journal 2012 Singapore, October of 22 University He* Accepted National Y. Sciences, Cynthia BioImaging ( for and correspondence Centre for Yuan NUS *Author Adam Sciences, Biological Y. of Wang, Department Chao Sun, Y. Stella in brucei morphogenesis cell to flagellum biogenesis links glycoproteins of adhesion consisting flagellum junction membrane intracellular An 520 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. npoylcform procyclic In lglu,Faelmatcmn oe(A) lglu deinpoen,Cl morphogenesis, Cell proteins, adhesion Flagellum (FAZ), zone attachment Flagellum Flagellum, .brucei T. and [email protected] Leishmania hc rlfrtsi h igtof midgut the in proliferates which , ) p.aeiprathuman important are spp. rpnsm brucei Trypanosoma Leishmania .brucei T. the , , is uhmlcl hrceie a P2 stage-specific, a GP72, The in was glycoproteins. protein characterized membrane membrane-associated glycosylated molecule via such body first cell the to 2011). al., unknown. et remains Zhou flagellum/ the assembly coordinated 1989; in the FAZ Gull, of of mechanism role and molecular Sherwin the membrane organization However, important 2003; plasma al., the the an et subtends (Kohl regulating plays that microtubules also by al., subpellicular et It morphogenesis, (Absalon 2010). bodies cell basal Vaughan, associated the including the 2007; organelles for and and single-copy required kinetoplast of 1989). the is segregation microtubules Gull, assembly ordered and highly flagellum/FAZ filament, (Sherwin coordinated reticulum The protein endoplasmic intracellular associated containing mostly a new flagellum 1999), new al., the structure a et (Kohl of of (FAZ) assembly zone Elongation the old attachment with 2001). the new accompanied as al., is the pattern flagellum et helical flagellum, whereas and (Moreira-Leite old length flagellum, the structure same along exact old elongates the FC, the duplicating the by at guided occurs flagellum, growth little lglaaddatclycagdcl opooy(opre al., et the (Cooper morphology FLA1, cell 1993). changed drastically null and GP72 flagella While 1996). viable, al., are the mutants et to non- (Haynes adjacent live, parasites on flagellum permeabilized immunofluorescence the indirect using along region detected adhesion readily was GP72 ntyaooe,temmrn-on lglu adheres flagellum membrane-bound the trypanosomes, In .brucei T. .cruzi T. Trypanosoma el ihu P2cnandetached contain GP72 without cells ooo oG7,i nessential an is GP72, to homolog rpnsm brucei Trypanosoma .cruzi T. Coe ta. 1991). al., et (Cooper rpnsm brucei Trypanosoma eerhArticle Research . Journal of Cell Science muolte ihat-F rat-L1 h seikmrsedgnu L1poenadarw akYP:L1 YFP::FLA1 YFP::FLA1, mark arrows and protein FLA1 endogenous YFP::FLA1 marks asterisk The anti-FLA1. YFP::FLA1 or anti-GFP with immunoblotted xrsigYP:L1 YFP::FLA1 YFP::FLA1, expressing rd eel F eotrdsrbto ttecl ufc.Aliae eeaqie n rcse sn h aecniin.( conditions. same the using processed and acquired were images a All indirect surface. whereas reporter, cell YFP the each at of distribution distribution cellular reporter overall YFP the reflects reveals (yellow) (red) fluorescence YFP Direct anti-GFP. with immunolabeled ebaeahso ewe lglu n el hsunique This cell. membrane– and the specific on for flagellum FLA1 basis between The molecular and adhesion the flagellum provides membrane FLA1BP. the is body, on cell domain the FLA1BP protein, extracellular between the a interaction FLA1-interacting with FAZ, interaction its the through novel attachment to flagellum for FLA1 important correct of for required is targeting tail 16-amino-acid the While characterized. S)a h -emns nNLrpa-otiigetaellrdmi ED,adatasebaedmi T)floe yaCtria 16-amino-ac C-terminal a by followed (TM) YFP::FLA1 ( domain (B), transmembrane residues. a YFP::FLA1 acid and amino expressing (ECD), the domain indicate extracellular Numbers NHL-repeat-containing (C16). an N-terminus, the at (SP) ihalreetaellrdmi n 16-amino-acid a mediates FLA1 and clear. how yet 2002), not domain is al., adhesion protein et extracellular membrane transmembrane flagellum (LaCount a large As tail cell 1996). cytoplasmic a al., (LaCount and et assembly Nozaki with attachment FAZ 2002; of flagellum al., regulation et both through likely in division, functioning protein, i.1 oanognzto fFLA1. of organization Domain 1. Fig. procyclic in protein nti td,w efre oanaayi fteFLA1 the of analysis domain performed we study, this In D D 1R a oe hnta fteedgnu L1 ossetwt hi eaieywa F nest hw nB n E. and D B, in shown intensity YFP weak relatively YFP::FLA1 YFP::FLA1, their of with expression consistent the FLA1, that endogenous Note the raised. of was that anti-FLA1 than which lower against was domain C16RR extracellular the contained that C16RR .brucei T. hr h ucino L1i best is FLA1 of function the where D C,YFP::FLA1 ECD, D ( C C,YFP::FLA1 (C), ECD A ceai iwo h oanognzto fFA n h rnainmtns L1cnan inlpeptide signal a contains FLA1 mutants. truncation the and FLA1 of organization domain the of view schematic A ) B–G D elsraeimnfursec nlsso F:FA n t rnainmtns ieclsstably cells Live mutants. truncation its and YFP::FLA1 of analysis immunofluorescence surface Cell ) 1,YFP::FLA1 C16, D 1 D,YFP::FLA1 (D), C16 D 1R,YP:L1T rYPaoewr rcintdb D-AEand SDS-PAGE by fractionated were alone YFP or YFP::FLA1-TM C16RR, sebyo lglu n A,admdae flagellum mediates and Results FAZ, coordinated and morphogenesis. the cell flagellum of facilitates regulation of junction assembly membrane intracellular aiu rtisicuiggot euaosadmembrane and regulators C- growth in found glycosylated, including a been have proteins which and a repeats, domain various NHL extracellular contain domain The to by predicted 1A). transmembrane was (Fig. tail followed a 16-amino-acid terminal peptide, domain, flagellum an contains for extracellular signal It required 2002). al., dually et N-terminal (LaCount glycoprotein cytokinesis surface and attachment a region is attachment flagellum FLA1 the mediate to can targeting domain, protein transmembrane FLA1, the of with domains together intracellular or extracellular Either D lglu deinpoen in proteins adhesion Flagellum 1R E,YP:L1T F rYPaoe()wr surface were (G) alone YFP or (F) YFP::FLA1-TM (E), C16RR H yae fclsstably cells of Lysates ) .brucei T. D 1 or C16 t-F labeling nti-GFP D 1 or C16 dtail id 521 Journal of Cell Science fRA-eitn L1mtns hc eete xrse in of depletion expressed tetracycline-inducible The then 2). were (Fig. which cells FLA1-RNAi construction mutants, the FLA1 for template RNAi-resistant as of used and material synthesized (supplementary was S3) sequence Fig. coding FLA1 scrambled this, achieve with To cells body. cDNA cell in the expressed from were detached could mutants flagellum mutants FLA1 with the FLA1 if the distinguished of be localization only precise the that could reasoned domain, region. adhesion transmembrane flagellum the the to with targeting protein 16-amino-acid together mediate C-terminal FLA1, the of or beads, that tail suggested domain extracellular results results streptavidin the These the either verifying assays. with immunofluorescence not, the could purified of alone YFP affinity and YFP::FLA1 (supplementary YFP::FLA1-TM and YFP::FLA1, assays furtherbiotinylated Whereas biotinylation was S1). The YFP::FLA1 reporters surface Fig. 1F,G). YFP (Fig. material by region the adhesion of evaluated flagellum accessibility the specific extracellular no to showed but intracellularly targeting flagellum detected readily however, the both alone, were reporter into transmembrane YFP the and (YFP::FLA1–TM) incorporated to only fusion domain YFP stop be 1C–E). as (Fig. all region domain attachment could transmembrane signal), (YFP::FLA1 the transfer after tail immediately inserted 16-amino-acid YFP::FLA1 truncation C-terminal FLA1 (YFP::FLA1 domain Interestingly, the extracellular 1996). the with al., lacking mutants probed et were (Haynes cells and non-permeabilized anti-HA the live, along localization found when also was flagellum FLA1 GP72 HA-tagged was Similarly, 1996). on al., This et reports in region. topology previous attachment membrane region with flagellum reporter attachment YFP the consistent the flagellum of at presence exclusive extracellularly the the confirming along 1B), (Fig. these cells in labeled YFP::FLA1 folding non-permeabilized poor specifically of or probing 1H) Anti-GFP (Fig. The overexpression cells. pathway. YFP secretory to also the may due system be endomembrane into the entry in observed signal its YFP peptide allowed strong signal that N-terminal the FLA1 of in presence the with observation consistent This was shown). not overlapped (data that reticulum endoplasmic cell in the with parasite found the were throughout peptide, structures signal specific reticulated the with mutants, after allowed FLA1 immediately cells All inserted surface. intact YFP cell at of reporters all YFP labeling of reporters anti-GFP detection YFP cell, of the distribution over the reflected indirect fluorescence direct or Whereas YFP 1B–G). fluorescence (Fig. YFP anti-GFP using direct immunofluorescence in monitored by then cells was non-permeabilized mutants procyclic FLA1 YFP-tagged in of localization overexpressed stably constructed were and fusions YFP various protein, this of requirements adhesion. membrane flagellum/cell the mediated FLA1 the adhesion how whether flagellum or clear only in membrane the not characterized cell Being protein was the both. it with or But associated membrane flagellum 1996). was al., GP72) et (or (Haynes FLA1 Nozaki region adhesion 1996; flagellum 1998). al., the et like Ruvkun, along FLA1, present and that was Slack GP72, showed 2003; analyses immunofluorescence al., protein–protein Previous et for required (Edwards be to interactions shown been have and receptors 522 u otefaelmatcmn ln h elbd,we body, cell the along attachment flagellum the to Due targeting the and FLA1 of localization precise the examine To ora fCl cec 2 (2) 126 Science Cell of Journal D D 1 n YFP::FLA1 and C16 were residues arginine two which in C16RR, .brucei T. .brucei T. LCute l,20;Nozaki 2002; al., et (LaCount hsfr twsas o clear not also was it far, thus D 1R ol l be all could C16RR .brucei T. Fg ) The 1). (Fig. D D 1 and C16 C)or ECD) D ECD, fedgnu L1adteidcbeepeso fYFP::FLA1 of expression inducible ( the FAZ. and the FLA1 along endogenous side of body cell the on exclusively YFP::FLA1 elbd Fg A.I hs el,teRNAi-resistant the cells, these In the 2A). from (Fig. detachment flagellum YFP::FLA1 body to was leading FLA1 cell endogenous 2B), (Fig. the of addition FLA1-RNAi, depleted the induce upon to 2002), al., tetracycline with et Consistent (LaCount 2B). observed (Fig. respectively previously anti-GFP, and (Nozaki 1996) FLA1 al., against et antibody monoclonal a using by immunoblots monitored were mutants truncation FLA1 YFP-tagged resistant, RNAi- of expression tetracycline-inducible and FLA1 endogenous ctee oswr on ntesd ftecl oy(rohas.In (arrowheads). body cell the of side the YFP::FLA1 few on a contrast, (arrows), found pattern were dotted a dots in scattered flagellum detached the to primarily targeted i.2 h C n h 1 nFA ahmdaeseii agtn to targeting specific mediate region. each FAZ FLA1 the in and C16 the flagellum and the ECD The 2. Fig. h yohtc L1bnigpoeno h lglu membrane. flagellum identify the on to protein FLA1-binding employed hypothetic, the was approach co-immunoprecipitation FLA1BP protein, An FLA1-interacting an of Identification DAPI and YFP::FLA1 (red) FAZ Whereas for BSA (blue). L3B2 3% (green), DNA with PFA anti-GFP for blocked with 4% minutes, labeled with then 5 and fixed another PBS for were methanol in Cells cold and body. minutes cell 5 the for led from which FLA1-RNAi, detachment for flagellum induced to cells in expressed inducibly were mutants niGPeey2 or vrtecus finduction. of course control. the loading over hours 24 every anti-GFP akdwt 32atbd Kh ta. 1999). al., et (Kohl antibody filament L3B2 FAZ the with with YFP::FLA1 overlapping line marked continuous distinct, ucaesrcue ln h eahdfaelm oescattered YFP::FLA1 Some of flagellum. detached labeling the along structures punctate ihafaelmmmrn rti,tu mislocalizing thus protein, along membrane body flagellum cell specifically the interact YFP::FLA1 may a to domain amino anchoring extracellular 16 simplest with the for C-terminal and the required the FAZ, the that but be is may observations, results acids these these of for interpretation be explanations may There 2A). other (Fig. FAZ L3B2-labeled the with aligning side, D ECD D D D ECD C16 C16 R eemntrdb muoltigwt niFA and anti-FLA1 with immunoblotting by monitored were R R D R nteohrhn,lclzdmil in mainly localized hand, other the on , ECD otefaelmi ucaedots. punctate in flagellum the to oaie xlsvl otecl oisi a in bodies cell the to exclusively localized R D lcigteetaellrdmi)localized domain) extracellular the (lacking C16 R a lofudo h elbody cell the on found also was ( D A C16 NirssatFA truncation FLA1 RNAi-resistant ) R lcigtectpamctail) cytoplasmic the (lacking B h nuil depletion inducible The ) a Tblnwsue as used was -Tubulin D C16 R and Journal of Cell Science aee ihbt niGPadat-itn(i.3C,D), (Fig. anti-biotin and a YFP::FLA1 anti-GFP as the used both was representing with which a only, was labeled One YFP 3B). (Fig. expressing control negative cells to relative ad eeseiial eetdi YFP::FLA1 in detected then Two 3B–D). specifically were (Fig. beads immunoblots were and streptavidin bands staining the silver from by analyzed eluted Proteins with co-immunoprecipitation beads. streptavidin-coated for centrifugation- with purification affinity used extraction, by followed were anti-GFP detergent lysates and cell cleared sonication with lysis sotie nFg A L1RA el xrsigYFP::FLA1- expressing cells FLA1-RNAi D 3A, Fig. in outlined As neato ewe L1adFAB a lovrfe by verified The also 4A). transmembrane (Fig. was FLA1BP tail a and 40-amino-acid FLA1 C-terminal by between a interaction followed and domain domain NHL- domain, an a possessed Following FLA1BP a N-terminus, repeat-containing proteins. FLA1BP. the lack two near revealed the these peptide despite named signal between FLA1, similarity FLA1BP of sequence we that of to and of similar which Tb927.8.4050 strikingly organization analyses 1), genes Table identical uncharacterized Bioinformatic see two previously by Tb927.8.4100; single, (encoded a protein an identified Mass protein. was FLA1-binding spectrometry a other representing possibly The anti-GFP, by analysis. spectrometry , mass by confirmed C16 0kabn hc ol edtce nyb niboi,btnot but anti-biotin, by only detected be could which band kDa 80 R eeboiyae olblalsraepoen.Atrcell After proteins. surface all label to biotinylated were D C16 R , 1 D rti htwas that protein kDa 110 rti n hswas this and protein D C16 R cells h xrclua oano L1P sYFP::FLA1BP as require FLA1BP, not of pattern did pattern dotted domain localization a extracellular This in the 4B). flagellum (Fig. detached anti-GFP the by along only detected FLA1. like just the extracellular, including also domain, was transmembrane repeats, the NHL and peptide between YFP::FLA1BP signal region 4B). in localized the the (Fig. moiety that YFP supporting region extracellularly, the YFP::FLA1BP accessible that was attachment indicated also anti-GFP, flagellum result This the with along By un- 4B). exclusively labeling (Fig. In YFP::FLA1 cell to the not. similar surface over fluorescence, all or non- YFP found direct of was FLA1-RNAi by YFP::FLA1BP labeling cells, for control anti-GFP induced induced or cells fluorescence then permeabilized was YFP the YFP::FLA1BP by before of Localization and evaluated reporter 4A). peptide (Fig. YFP signal repeats the N-terminal NHL the inserting after by immediately constructed was YFP::FLA1BP procyclic culture for in essential survival membrane not for is required but protein adhesion, flagellum a is FLA1BP iia ota bevdfrYP:L1P(i.4D). pattern (Fig. dotted YFP::FLA1BP a for in observed flagellum, that detached to similar the to properly targeted rcptt ihYP:L1P(upeetr aeilFg S4). Fig. co- material to (supplementary YFP::FLA1BP shown with was precipitate FLA1 where co-immunoprecipitation, reverse neetnl,i L1RA el,YP:L1Pwas YFP::FLA1BP cells, FLA1-RNAi in Interestingly, lglu deinpoen in proteins adhesion Flagellum sFLA1BP. as named protein FLA1-binding putative uncharacterized, previously arrow) a red identified (B, band weight lower molecular the of analysis spectrometry sda oto.( control. were a only as YFP used expressing Cell details). for Methods and Materials (see FLA1BP protein itnltd YFP::FLA1 biotinylated, identify to procedure purification purifications. affinity and (co-IP) co- immunoprecipitation sequential binding by FLA1 FLA1BP, a protein, of Identification 3. Fig. eonzdb niGP()adtherefore YFP::FLA1 and represented (C) anti-GFP also by was recognized band weight The molecular (D). higher labeling as anti-biotin biotinylated by were shown proteins both and (B) staining silver were by bands detected protein specific two process, purification co-IP/affinity the sequential After products. co-IP the of immunoblots ( A lwcato the of chart Flow ) .brucei T. B–D ivrsann and staining Silver ) D D .brucei T. C16 C16 R R Mass . binding D ECD 523 Journal of Cell Science eeantto eeIDs Gene annotation Gene 524 eltdo F2(i.4)(atne l,19) ugsigthat axoneme. suggesting flagellum 1998), with al., directly additionally et interact (Bastin were dotted may 4E) that FLA1BP its (Fig. PFR PFR2 nor lacking of cells flagellum, depleted in the affected was to pattern the YFP::FLA1BP Neither repeats. of paraflagellar structural regular a localization with and both axoneme lattice, (PFR) microtubular rod 9+2 skeleton, a flagellum contains to FLA1BP which of anchoring possible a suggested FLA1BP FLA3 FLA2 FLA1 Hypothetical Hypothetical eoe ako infcn eunesimilarity. sequence significant of lack denotes X h otdlbln atr fFAB ln h flagellum the along FLA1BP of pattern labeling dotted The ora fCl cec 2 (2) 126 Science Cell of Journal b2.068 ohYsYes Yes Both X X X Tb927.10.6180 Tb927.8.4050; Tb927.5.4570; b2..005%7.%10 S e Yes Yes Yes Yes Yes Yes BSF BSF Procyclic X X X 100% 60.6%/72.8% 97.5%/98.3% 60.6%/72.8% 100% 58%/71.1% Tb927.8.4110 Tb927.8.4060 Tb927.8.4010 Tb927.8.4100 Tb927.5.4580 al .NLrpa-otiigtasebaepoen in proteins transmembrane NHL-repeat-containing 1. Table L1FA FLA1BP FLA2 FLA1 0%PoylcYsYes Yes Procyclic 100% X X 05/49 S oNo No BSF 40.5%/54.9% X X rmr euneidentity/similarity sequence Primary Fg BC.A pcfcatbd gis L1Pwsnot monitored was was induction tetracycline FLA1BP upon depletion its against FLA1BP-RNAi and in cells antibody expressed stably was specific YFP::FLA1BP available, RNAi As immunoblots Efficient and 5B,C). (RT)-PCR RNAi. (Fig. transcriptase reverse inducible by the verified using was mediate depleted in could was interaction observed FLA1BP their adhesion that flagellum–cell suggested FLA1 with h rsneo L1Po h lglu n t interaction its and flagellum the on FLA1BP of presence The L1RA el tbyepesn F:FAB eeused (blue). were control. DNA YFP::FLA1BP as for expressing DAPI stably and cells (red) FLA1-RNAi YFP::FLA1BP PFR monitor for cold to anti-PAR with anti-GFP (green), followed with PFA stained 4% and with methanol, fixed and were FLA1 flagellum. both Cells of the PFR2. depleted to cells localization in expressed FLA1BP was for YFP::FLA1BP required not ( is flagellum. PFR the its to revealed primarily labeling localization expressed Anti-GFP transiently cells. and was FLA1BP-depleted FLA1BP-RNAi depletion, in for RNAi used to region resistant the therefore contain not ( did YFP. which cells of 2). transfected levels Fig. all detectable (cf. of expressing FAZ representative the are along shown probably was Images surface, YFP::FLA1 cell FLA1BP, the of on depleted un- present cells in In region cells. adhesion flagellum induced the to showed localization antibody YFP::FLA1 Surface anti-GFP not. with or staining tetracycline immunofluorescence with anti- induced by cells flagellum FLA1BP-RNAi detached the ( of labeling. surface GFP the on detected YFP::FLA1BP was cells, FLA1-depleted in whereas flagellum region, the In adhesion along Methods. detected and was YFP::FLA1BP Materials using cells, the uninduced examined in was and described PFA, as was 4% methods with labeling fixed Surface cells not. on of or performed absence RNAi or induce presence to the tetracycline in cultured cells FLA1-RNAi F uin,teYPwsisre meitl ontemof downstream ( immediately peptide. inserted signal was the generate YFP To the tail. fusions, 40-amino-acid YFP a a and domain, domain NHL-repeat-containing transmembrane an with signal followed N-terminal (SP) an peptide contains YFP FLA1BP and FLA1, FLA1BP to of Similar organization fusions. domain the its of and domain. view extracellular schematic protein the membrane of flagellum independent is a localization is FLA1BP 4. Fig. RAexpression mRNA profile C F:FA a rninl xrse in expressed transiently was YFP::FLA1 ) .brucei T. B F:FAB a tbyepesdin expressed stably was YFP::FLA1BP ) Leishmania .brucei T. D YFP::FLA1BP ) E h rsneo a of presence The ) Orthologs spp. ots this, test To . .cruzi T. D ECD, ( A )A Journal of Cell Science ulu n h otro n ftecl a 6.3 was (7.1 cell cells control the in of that than end slightly posterior only decreased the and nucleus lglu eghadFZlnt r togycorrelated strongly are length FAZ and ( length flagellum rmtekntpatadascae aa oist h posterior the distance to the bodies hand, basal the other associated in the and shortening kinetoplast On to the parasites. due from the mainly of was region body anterior cell the of 9.2 shortening from FAZ), the shortened 6.6 of presence is of the tip by however, anterior marked the region and (anterior nucleus parasite the the between distance The 6C). Fig. 5.8 ftecnrlcls(13.7 cells control the of oeta w pcfcbnswr aee ihat-F nYP:L1Pepesn el.Bt ad ol eimnpeiiae ihat-F (see anti-GFP FLA1BP-RNAi. with of induction. immunoprecipitated expression RNAi be upon of could observed course bands was the Both bands over cells. both hours YFP::FLA1BP-expressing of 24 in depletion every anti-GFP and anti-GFP with (B S4) ( using levels labeled Fig. days. monitored mRNA were FLA1BP material 7 was for bands supplementary after YFP::FLA1BP analysis specific even of RT-PCR two and control degradation (A), that un-induced The analyses the Note cells. growth FLA1BP-RNAi to for hours rate in 24 expressed similar every a stably taken at were samples duplicated and cells RNAi, FLA1BP-depleted induce to tetracycline of presence or eue rsial rm12.1 from drastically reduced i.6.Tefaelm ept en eahdfo h elbody, cell the 14.3 from at detached measured being despite in the flagellum, summarized The were to 6C. cells parasite Fig. relative the of positioning tips length), anterior nucleus/kinetoplast and flagellum posterior of and indicator length (an FAZ length Detailed PFR post-induction. days of 7 measurements at the population asynchronous of the represented of which disruption flagellum, single a to a and nucleus, kinetoplast single single due a contained we that likely this, cells test FLA1BP-depleted analyzed To were 6A,B). (Fig. which assembly flagellum/FAZ positioning coordinated length, organelle cell in changes and morphological also cells drastic FLA1BP-depleted showed detachment, flagellum FAZ to and addition flagellum In the the of for assembly required coordinated are adhesion. proteins adhesion flagellum Flagellum in FLA1BP of role cell the supporting from post- 6), detached days Fig. in Flagellum 7 exhibited morphology. even in cells body defects control in FLA1BP-RNAi un-induced changes However, obvious the drastic 5A). to (Fig. show the similar duplicate induction to rate depletion, continued not a FLA1BP at lacking FLA1BP did Cells rate. efficient proliferation cells Despite FLA1BP-RNAi anti-GFP. using culture. in survival cell procyclic for essential not is FLA1BP 5. Fig. L1PRA el ( cells FLA1BP-RNAi R 2 hr A e osotcl egh h itnebtenthe between distance The length. cell short to led FAZ Short 6 6 5 1.6 1.5 .5,ti orlto a opeeyaoihdin abolished completely was correlation this 0.75), . m m 0 fteclsosre t2dy otidcin(cf. post-induction days 2 at observed cells the of 90% nFAB-bae cells. FLA1BP-ablated in m nFAB-Nicls(i.6) ugsigthat suggesting 6C), (Fig. cells FLA1BP-RNAi in m 6 2.3 m nFAB-Nicls iia othat to similar cells, FLA1BP-RNAi in m R 6 2 1.9 5 .1 i.6) h A length FAZ The 6D). Fig. 0.21; m ) hra ncnrlcls the cells, control in Whereas m). 6 6 2.1 1.7 m ncnrlclsto cells control in m m ncnrlclsto cells control in m 6 6 1.4 1.8 , 60% m ( m A m; m, , B el ihasal nertdFAB-Nicntutwr rw nteabsence the in grown were construct FLA1BP-RNAi integrated stably a with Cells ) Fg C,tu eutn ntekntpatlctdslightly located kinetoplast the in resulting thus 6C), (Fig. hr e A n e lglu sebe nahighly a in assembled flagellum new ( and and control fashion FAZ cells both coordinated control old in in new structure, the unlike old However, where to the 7A,B). to (Fig. attached cells attached tip FLA1BP-RNAi tip distal distal FAZ, old it its the along with extended also with FAZ new the FC, flagellum, the via flagellum old assembled was the flagellum new was along the duration Whereas cycle 5A). cell in Fig. change (cf. observed significant as no rate and same cells the control at the proliferated cell cell FLA1BP-RNAi each that theobservation of with both consistent timing was In This and unaltered. largely 7). remained order event (Fig. cycle the cells, cycle FLA1BP-RNAi cell and the control throughout cells examined were as FLA1BP-RNAi flagellum new kinetoplast, and in and FAZ associated duplication new of and assembly the the bodies as including well basal events nucleus, cycle of segregation cell major FAZ, short 6A–D). (Fig. morphology cell length, normal FAZ as and well length as flagellum flagellum between restored in correlation removal expression adhesion, FLA1BP tetracycline region Restoring by FAZ 4C). cells FAZ the (Fig. FLA1BP-RNAi side to body the localized cell properly the to Fig. on also material addition was supplementary 6A; YFP::FLA1 In (Fig. S5), FAZ1 cells. and CC2D FLA1BP-RNAi markers 6A). in Fig. assembled (see cells FLA1BP-RNAi in nucleus the to anterior i,sgiiatyicesdfo 4.5 from increased significantly tip, lwdaduculdfo e lglu lnaini FLA1BP- in elongation ( cells flagellum RNAi new from uncoupled and slowed oiinn nduhe el c.Fg C.Ncerdivision, kinetoplast Nuclear in 7E). 6C). (Fig. Fig. change FAZ short (cf. the the by affected cells moderately only hence was daughter however, in 7D), in observed (Fig. segregation positioning parasites body/kinetoplast basal these inhibited the for Mysie l,20;Wlo n eeve 04,b niiinof inhibition by 2004), Lefebvre, and Wilson 2009; al., et (Miyoshi flagellum/ lengthen in to cilium shown been has treatment chloride Lithium length regulation FAZ flagellum of mediate proteins adhesion Flagellum ofrhrudrtn o L1PRA el iie iha with divided cells FLA1BP-RNAi how understand further To normally appeared FAZ the length, short the Despite lglu deinpoen in proteins adhesion Flagellum hayooa reinhardtii Chlamydomonas C R omntrFAB rti elto,YP:L1Pwas YFP::FLA1BP depletion, protein FLA1BP monitor To ) 2 5 .5 i.7) h hr A ol loaccount also could FAZ short The 7C). Fig. 0.35; R 2 5 .2,eogto ftenwFZwas FAZ new the of elongation 0.92), n oemmaincells mammalian some and 6 1.0 .brucei T. m mto6.8 6 1.6 525 m m ). Journal of Cell Science mn cd n ilclzdt h lglu ebae we membrane, flagellum the 16 to C-terminal mislocalized the and lacked acids that mutant amino FLA1-truncation a Using the Discussion to 8C, information (Fig. length length flagellum FAZ FAZ. intracellular transmitting in in difference adhesion show not P did 8B, and FLA1BP (Fig. intact control where was cells adhesion control both flagellum in observed only in was however, increase, 8B,C, length length (Fig. flagellum cells a in FLA1BP-RNAi of resulted treatment LiCl increase The 6. then parasite significant Fig. one were in and on described kinetoplast lengths one as nucleus, flagellum, effects FAZ one containing and inhibitory cells in Flagellum measured for little LiCl 8A). mM (Fig. showed 5 proliferation of which absence or presence hours, the 24 in cultured were to not assembly or FAZ linking in role elongation, flagellum active an flagellum play that confirm proteins To adhesion (GSK3). 3 kinase synthase glycogen the 526 5 .5,spotn nesnilrl ffaelmmembrane flagellum of role essential an supporting 0.35), ora fCl cec 2 (2) 126 Science Cell of Journal .brucei T. el nue ihFLA1BP-RNAi with induced cells P , .01.SgiiatFAZ Significant 0.0001). P , .01.Clslacking Cells 0.0001). ec ikn h lglu ebaet elmembrane. cell to membrane axoneme. domains, flagellum extracellular the flagellar their linking to through hence interact anchored facing FLA1BP the and possibly acids spans FLA1 amino and hand, 40 other lumen assembly C-terminal cell the flagellum the FAZ on the with intracellular FLA1BP, in membrane, in 2002). acids flagellar al., role amino et a 16 (LaCount C-terminal playing membrane its possibly cell is with the lumen, spans adhesion FAZ FLA1 model, the flagellum–cell this along In mediates 9. Fig. interaction in interaction proposed this their of how model Co-immunoprecipitation A and domains. probably FAZ. extracellular FLA1BP, their and the FLA1 through distinct between along interaction a the to membrane confirmed revealed FLA1 and cell flagella, membrane flagellum and short the detached the to FLA1 with FLA1BP YFP-tagged of and localization cells similar of in analyses with have domains FLA1BP Further proteins in FLA1 tails. transmembrane similarity NHL-repeat-containing cytoplasmic and of are FLA1-binding lack both FLA1BP extracellular, first the organization: sequences, the Despite domain acid is date. which to amino FLA1BP, identified novel protein a isolated orltdt lglu egh( length flagellum to the correlated of in center measurements the length to or ( distance cell are the values of All tip nucleus. and anterior between kinetoplast or distance the and posterior to dot), the cell green (small the bodies of basal tip associated between anterior distance or length, posterior were FAZ the line), length, (green flagellum FAZ for and quantified blue line) (small (red kinetoplast flagellum circle), circle), blue (large nucleus single n L1PRA eesdcls( cells reversed FLA1BP-RNAi and a irpe nFAB-Nicls( cells FLA1BP-RNAi in disrupted was otyrsoe neFAB-Niwsreversed was ( FLA1BP-RNAi once restored mostly h el eefxdwt odmtao n aee ihanti- with labeled and methanol cold with For fixed (B). were cells microscopy the electron A scanning for or fixed (A) were immunostaining cells FLA1BP-RNAi-reversed and ( RNAi hours. 48 tetracycline-free for in by medium culture followed continuous and and off days wash 7 tetracycline for tetracycline flagellum with for induced crucial morphology. is cell FLA1BP and of adhesion expression The 6. Fig. el nec ae r eactdb ht otdlines. dotted white by demarcated Two are ( (blue). panel DNA each the for in of DAPI cells most and along (red) present flagellum was extracellular for that anti-PAR rod (green), FAZ paraflagellar and the (arrows) bodies basal for CC2D D R C 2 nu-nue oto el,teFZlnt a strongly was length FAZ the cells, control un-induced In ) nidcd( Un-induced ) 5 .8 green). 0.68, n 5 0 el) L1PRA ( FLA1BP-RNAi cells), 109 A , B nidcdcnrl FLA1BP- control, Un-induced ) R 2 5 L1PRA el were cells FLA1BP-RNAi .5 le.Ti correlation This blue). 0.75, n 5 0 el)cnann a containing cells) 100 R 2 5 .1 e) and red), 0.21, n 5 9cells) 99 m m. Journal of Cell Science irpe,drn ohcl yl eeomn n LiCl and the normally, development was formed cycle flagellum assembly new cell flagellum the flagellum/FAZ Although and both treatment. FLA1BP coordinated during lacking disrupted, cells the In adhesion, membrane. to adhesion attachment cell membrane flagellum the of basis membrane molecular exact the provided flagellum the of molecular the dissection required structures. assembly Further of al., et coordinated organization 2011). Vaughan characterization the al., 1995; of al., microtubule et mechanisms et organelle Zhou Robinson subpellicular 2002; division, 2008; al., cell et an and is (LaCount for which FAZ, important segregation the the complex with via flagellum is intracellular cell of function regulates assembly latter also This coordinated it 2010). functions, (Vaughan, sensory morphogenesis and motility to addition ( oeaeyihbtdi L1PRA cells. FLA1BP-RNAi in inhibited moderately In adhesion membrane flagellum/ cell requires assembly flagellum/FAZ Coordinated fl cells, control uninduced dist the its in Whereas with (E). also nuclei structure, two FAZ or old (D) the ne kinetoplasts to a two posterior (B), (C), assembled with flagella cells kine was two coordinately associat FLA1BP-depleted of FAZ containing remained elongated In number flagellum new cells FAZ panel. the new in A each the on measured FC. of of were based intact tip right nuclei progress the distal ( dividing the cycle through the structure. on probably detached, cell old shown were it, the of flagella and to with order both attached traced along the Though segrega also elongated flagellum. in and were and old presented duplication cell flagellum the the are to old each by Images next in accompanied assembled dots). PA organelles new was was blue a (green), FAZ major flagellum and (large FAZ new detached These flagellum and and nuclei flagellum. new flagellum bodies the a and new basal (carets), and FAZ the bodies for dots) nucleus, basal of anti-CC2D the blue elongation with of coordinated (small immunolabeled duplication The kinetoplasts and following structures. (A), fixed the old cells were control the hours un-induced to In 72 posterior (blue). for assembled DNA tetracycline for of DAPI assembly. presence and (red) flagellum/FAZ or flagellum absence coordinated the for in essential cultured is cells adhesion Flagellum 7. Fig. R 2 h dniiaino L1P nitrcigprnro FLA1, of partner interacting an FLA1BP, of identification The 5 .brucei T. .5.Atog ieols ergto a infcnl niie n o oriae ihFZeogto,tesgeaino h uliwsonl was nuclei the of segregation the elongation, FAZ with coordinated not and inhibited significantly was segregation kinetoplast Although 0.35). h lglu samliucinlognle In organelle. multifunctional a is flagellum the , C–E R h egho h e A n e lglu,tgte ihtedsacsbtensgeaigkntpat/aa oisand bodies kinetoplasts/basal segregating between distances the with together flagellum, new and FAZ new the of length The ) 2 5 .2 nteFAB-Nicls h A sebysoe,adwsntcodntdwt lglu elongation flagellum with coordinated not was and slowed, assembly FAZ the cells, FLA1BP-RNAi the in 0.92; lglu–elahso n h oriae flagellum–FAZ coordinated the and adhesion flagellum–cell basal of associated the segregation proper and for the on nuclei. responsible kinetoplasts effect the moderate also the only the had be the was but of bodies, that in FAZ may segregation microtubules short region suggesting limited The subpellicular posterior region. the region, anterior to the differently anterior in regulated the microtubules subpellicular in culture. in particularly least at survival, cell and division cell segregation, nrni eghrglto fcr A opnns oee,it an However, or a components. below), FAZ that core (see appeared of proteins regulation FLA1-related length or other intrinsic FLA1BP, of of depletion presence incomplete the to due was assembly partial bevdi oto el (12.1 cells control in was observed FAZ short This markers. 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GP72 and on body work Previous cell cruzi morphology. the cell to the normal transmitted in restoring efficiently contained be information to length flagellum the allowing thus assembly, ( LiCl with treatment hours hours 24 24 every after monitored significantly was increased proliferation ( cell days. the 4 and over LiCl, mM 10 and in proliferation. growth parasite flagellum/FAZ coordinated for treatments. required LiCl is FLA1BP 8. Fig. 528 rdcdsmlrrslsi elmrhlg Coe tal., et (Cooper morphology cell in results similar produced ora fCl cec 2 (2) 126 Science Cell of Journal B ncnrlprsts ohfaelmadFZlengths FAZ and flagellum both parasites, control In ) ( A ramn ihu o5m ilhdltl feton effect little had LiCl mM 5 to up with Treatment ) .brucei T. 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FLA1 either encoded to to homology is no database, similarity shows protein the 54.9% FLA1-like and in by sixth identity (encoded found protein 40.5% was While by FLA1BP-like showing Tb927.5.4580) a FLA1. and FLA1, shows (encoded to Tb927.5.4570 Tb927.8.4100) to and respectively, similarity FLA3 Tb927.8.4050 little similarity, by (encoded 70% and FLA2 FLA1BP and of FLA1BP sequences Tb927.8.4060) identity and acid are FLA1 amino by Tb927.8.4110) as the fashion them, (encoded Among similar NHL 1). in and (Table domains arranged transmembrane repeats containing proteins encode In proteins FLA1-related The h deinbtentefaelmmmrn n h cell the and membrane flagellum the between adhesion The .brucei T. aaae oradtoa ee eepeitdto predicted were genes additional four database, D C.WeesFAB-eltdclswere cells FLA1BP-depleted Whereas ECD. . 7 dnia,adec exhibits each and identical, 97% , 60% Journal of Cell Science 06b rgeto L1(uloie 417)(aon ta. 2002) mutant al., with resistant created FLA1-RNAi et of line expression cell (LaCount the RNAi for 4B). used 64–1070) YFP::FLA1 Fig. was 2A; and fragment (nucleotides Fig. 302-bp growth S2; the FLA1 cell Fig. a with material of on observed (supplementary previously effects fragment phenotypes RNAi inhibitory the 1006-bp produced to similar constructs attachment, tetracycline-inducible flagellum RNAi allows which in Both resistance, nucleotides blasticidin RNAi. with 112 2000) and al., et sequence (Wang coding the fragment of 390-bp 3 1364–1641 a or (nucleotides 715–1016) FLA1 (nucleotides of fragment 302-bp a FLA1-RNAi, methods For transfection and construction Plasmid in cultured was 1983) al., bovine et fetal 28 heat-inactivated (Ruben at 15% line (Hyclone) with cell supplemented serum medium YTat1.1 Cunningham’s form Procyclic culture Cell Methods and Materials to similar Each 1). membrane (Table spp. including parasites flagellum–cell kinetoplastid other in the the substantiates of development. during specificity, adhesion complexity distinct FLA1- developmental but six organization remarkable of and suggested domain presence in similar The studies sequences development with 2011). proteins, al., Previous parasite et related (Rotureau during parasites. flies remodeling BSF tsetse FAZ in FLA1BP- significant with protein interact may FLA2/3 like elongation. whereas cells, flagellum membrane procyclic that from in assembly flagellum–cell speculate FAZ adhesion mediates uncoupling to body, flagell tempting interaction cell the the is of FLA1–FLA1BP from assembly it detached the coordinating is proteins, thus flagellum FLA1BP-like respectively, the axoneme, FLA1BP, t membra flagellum or body, cell and adhe FLA1 the cell FAZ membrane on either the flagellum the present of mediating from with is thus absence exit FLA1 interact domains, the model, Upon may extracellular this In the (FPC). FLA1BP In through complex. and collar adhesion. interact FAZ FLA1 pocket FLA1BP this and of for flagellar FLA1 essential tails the membrane. are cytoplasmic flagellum by FLA1BP The the membrane and on FLA1 cell present Both is the membrane. FLA1BP from cell and FLA1BP. partitioned the and to is FLA1 adheres membrane of membrane interaction flagellum flagellum the the by mediated and adhesion pocket flagellum flagellar of model A 9. Fig. erccieidcbeepeso Wrze l,19)wsmitie in maintained was 15 tetracycline- of 1999) 28 presence heat-inactivated, the at in hygromycin 15% al., (Clontech) serum bovine with fetal et free supplemented medium (Wirtz Cunningham’s expression tetracycline-inducible sntesnilfrnra elfntosas pn pnwways pathogens. new up human opens lethal also these functions possible combat cell to normal The for that body. essential proteins not surface cell is non-variant two to a between flagellum interaction in study specific from to junction opportunity transduction fascinating adherence signal a 2000). represents membrane al., parasite et intracellular unicellular (Weise FAZ unique short a This with attached partially only are 9 utasae ein a C mlfe n lndit ZMconstruct pZJM a into cloned and amplified PCR was region) -untranslated rhlg oms fteFA-eae rtisaeas found also are proteins FLA1-related the of most to Orthologs D C;RA elln rae ihte39b rgetwsue for used was fragment 389-bp the with created line cell RNAi ECD; .brucei T. ˚ C. lglain Flagella . ˚ .Poylcfr 91 elln nierdfor engineered line cell 29.13 form Procyclic C. .cruzi T. elcnan natce flagellum attached an contains cell Leishmania .cruzi T. m /lG1 n 50 and G418 g/ml aaie,however, parasites, and Leishmania m g/ml h xrsino NirssatYFP::FLA1 RNAi-resistant of expression the n 5 and osrce ypaigtogntccdn o riie()adaso codon stop a and (R) 5 arginine primers for with PCR codons through amplification 1519–1590) (nucleotides genetic domain transmembrane the two after immediately placing by constructed YFP::FLA1 GGATCCGTGTGTCTCGGTGGTATCGTTTCCTC-3 AGATCTGTGGGCACCAAGGTGATTGTGAACC-3 htcnandteNtria rnmmrn oanwsPRapiidwith amplified PCR was domain 5 transmembrane N-terminal primers the contained that S3B). Fig. material (supplementary respectively 3 ieudrie)ad5 and underlined) site F napD04vco ihprmcnrssac Qiaae l,20) the 5 2002); al., PCR-amplified et then primers was (Quijada 136–2253) resistance (nucleotides with sequence puromycin coding with FLA1BP vector of rest pHD1034 a in YFP with digested underlined), site lentvl,FAB oigsqec otiigol h C-terminal was the 2059–2250) only (nucleotides tail containing 5 primers 40-amino-acid with sequence amplified and PCR YFP::FLA1BP. generating coding domain thus YFP, transmembrane FLA1BP of C-terminus the Alternatively, to fused and underlined), nelnd n 5 and underlined) ietdwith digested GCT rmr 5 from PCR-amplified was primers which peptide FLA1, signal of the blasticidin-resistance 99-bp for N-terminal codes The with proteins. 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Hart, S., Griffiths, H., Farr, R., H. Dawe, R., Broadhead, Berriman,M.,Ghedin,E.,Hertz-Fowler,C.,Blandin,G.,Renauld,H., K. L. Gull, J. and T. Roggy, Sherwin, and P., Bastin, M. D. Ransom, M., E. Brouch, D., J. Bangs, unnhmsmdu iufr2husadatce ocvrlp by coverslips to with post- twice and attached water, washed distilled then with and time were O one 1% filtered), Cells hours 7.4, with minutes. 2 (pH fixed 10 buffer cacodylate for in for M rpm 0.1 glutaraldehyde 4000 2.5% situ at with centrifugation fixed medium were cells Cunningham’s microscopy, electron scanning For microscopy electron Scanning bao,S,Kh,L,Bace . lsik . otri,G,Rsoi . Cosson, F., Rusconi, G., Toutirais, T., Blisnick, C., Branche, L., Kohl, S., Absalon, References Research at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.113621/-/DC1 National online available Singapore material Supplementary the C.Y.H.] by to NRF-RF001-121 supported number [grant was Foundation work The Funding Philippe McMahon-Pratt and Diana Professor Gull and anti-PAR. antibody, Keith for the L3B2 Professor for the for Cross (anti-FLA1), Bastin George Professor for antibody thank Kabututu for Pius Flap2 also University Zakayi We Dr Technological assistance. and Nanyang technical spectrometry at mass Mei with Meng assistance Ms thank We Acknowledgements vprtr(EL,advee sn eisNnLbDaBa electron vacuum DualBeam a NanoLab by Helios carbon a After with using minutes. coated 20 viewed (FEI). dried, for microscope and twice point ethanol, ethanol (JEOL), critical 90% 100% evaporator ethanol, and were 80% minutes cells 10 ethanol, dehydration, for 70% each ethanol, ethanol 50% concentrations increasing 95% ethanol, in 30% dehydrated and ethanol: once of water distilled with washed were rpnsm rz lcpoenGp72. glycoprotein cruzi Trypanosoma cycle. life Leishmania the during function ufc ftrypanosomes. of surface P. Overath, and N. complex. repressor translation tumor-Pumilio brain 2508-2513. the of Model adhesion. flagellum-cell 122 of disrupts glycoprotein antigen surface stage-specific cruzi Trypanosoma insect an GP72, for gene cruzi. candidate Trypanosoma a of trypanosome. bloodstream Characterization the al. et of G. viability P. the McKean, for J., S. required al. Gaskell, is L., et M. B. Ginger, K., Haas, M. E., brucei. N. Trypanosoma Hamlin, trypanosome E., African Caler, the 416-422. of J., genome N. The Lennard, (2005). C., D. Bartholomeu, motility. reticulum endoplasmic on Studies brucei. Trypanosoma targeting. in system reporter secretory otoldb lglu omto nTyaooabrucei. Trypanosoma P. in Bastin, formation and flagellum D. by controlled Robinson, M., Bonhivers, J., 149-156. , Nature .Bo.Chem. Biol. J. S O 391 4 nH in 548. , 20) yrdnmcfo-eitdpoensrigo h cell the on sorting protein flow-mediated Hydrodynamic (2007). o.Bohm Parasitol. Biochem. 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