Journal of Cell Science 1 Ikeda N. Kyojiro inheritance flagellum for in necessary is kinase Polo-like Article Research aldcnrn n ecn-ihrpa rti nw as known protein repeat leucine-rich a proteins calcium-binding and small This centrins contains 2009). also termed al., called structure which cytoskeletal et larger bilobe, (Morriswood a the of FAZ part the is complex of MORN1 part with posterior coincides which the of MORN1- stem a the the of complex, pocket extension cytoskeletal flagellar hook-like containing the against the (Bonhivers of by neck encircled pocket compartment partially The also 2009). is the al., flagellar et Gadelha to the 2008; al., access et of limits neck which BILBO1, flagellum, the protein flagellar the clinches The comprising compartment. structure tightly a this (FPC), exit collar therefore pocket flagellar must the of and membrane the pocket from emerges flagellum The al., et 2009). for Lacomble site 2009; of sole Carrington, invagination and the (Field is an exo-cytosis which and of endo- pocket, flagellar base the the called at surface cell located the Vickerman, is 1999; body basal (Gull, The (FAZ) 1969). zone attachment known flagellum structure the the cytoskeletal to as underlying attached an is by flagellum membrane mitochondrial The plasma 1991). the key Gull, (Gull, is and nucleus the Robinson the which 1999; from separate kinetoplast, 1A; maintained is (Fig. the that aggregate to DNA anterior basal adjacent The shown). the is also are body towards manuscript this extends of in end described posterior and structures the near essential cell body is basal which a the flagellum, by nucleated single is The requires motility, 2010). for that Hill, (Engstler host 2007; protozoan its al., of flagellated response et immune the a evade to order is in motility It Africa. sub-Saharan brucei Trypanosoma Introduction proper their for necessary be might structures these flagellum. attached on words: and Key presence positioned its properly which duplication a that the quartet, of in suggesting blocks microtubule inheritance cell and these the flagellum, the segregation the of body as throughout towards the basal each migrates known in to position microtubules, defects localizes TbPLK causes of that kinase biogenesis. TbPLK set The of regulators of specialized series body. Depletion the a a cell migration. to its the with of localizes during to associates successively kinase flagellum then TbPLK the pattern the and transport duplicating. localization flagellum, of might are dynamic the attachment nucleates a they and which has as position body, TbPLK the structures basal and that regulate the show separation to that We localizes structures parasite centriole bilobe. kinase protist cytoskeletal the The from the as cycle. In cell ranging known cell midbody. the the structure cells, the during as cytoskeletal then locations mammalian centrin-containing and a cellular in poles of different spindle duplication events to the move to mitotic homologs progressing of kinase centrosome, brucei Polo-like the variety roles, at a these starting fulfill progresses, in cycle To role abscission. to important congression an chromosome play kinases Polo-like Summary 10.1242/jcs.101162 doi: 3173–3184 125, ß Science Cell of Journal 2012 February 14 Accepted ( correspondence for *Author 2 nvriyo ina etrfrMlclrBooy a .Prt aoaois r oras /,Ven,Austria Vienna, 9/3, Austria BohrGasse Vienna, Dr. 9/3, Laboratories, BohrGasse Perutz Dr. F. Laboratories, Max Perutz Biology, F. Molecular Max for Vienna, Center of Vienna, University of Medical University Biochemistry, Medical of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h igepl-iekns homolog kinase polo-like single the , rpnsm brucei Trypanosoma rpnsm brucei Trypanosoma 1 stecuaieaeto leigscns in sickness sleeping of agent causative the is [email protected] n hitpe .d Graffenried de L. Christopher and L,Faelm elcce A,Bslbody Basal FAZ, cycle, Cell Flagellum, PLK, , ) .brucei T. .brucei T. L TPK sesnilfrctknssadi eesr o h correct the for necessary is and cytokinesis for essential is (TbPLK) PLK u h pcfclctost hc ttresadisfntosaegeared are functions its and targets it which to locations specific the but , 2, * rti opee htgietenwfaelmt t final its to flagellum new process? this the organizes then, guide What, those position. that 2001). of sequence al., complexes in et duplication protein new coordinated (Moreira-Leite as the the body known requires The of cell this structure positioning All the a IV). the along by guides flagellum 1B, one which new is (Fig. old (FC), flagellum connector the membrane new flagella to the the linked be plasma that also then so is the flagellum FAZ, must new to flagellum a new tethered of 1B, the growth (Fig. to of bilobe coupled the elongation and FPC Further the II,III). of flagellum. duplication emergent the an duplicate, contains requires to now This needs pocket this flagellar 2010). neck, each al., that pocket et so flagellar Lacomble the 1989; existing reaches Gull, body, it the and Once alongside (Sherwin probasal I) flagellum the 1B, new (Fig. of a one of maturation growth the triggers by which initiated Flagellum biogenesis. is flagellar of biogenesis process This the the 2008). for Gull, of of implications and has inheritance partitioning Vaughan 2010; and proper (Vaughan, assembly complexes The these coordinated membrane FAZ). the plasma the requires the and of flagellum length bilobe collar the the pocket along flagellar (using and the bilobe) through FAZ, base and its (FPC the near flagellum, of the part of become 2008; 2009). al., then et (Lacomble al., and structures the flagellar FPC all the connecting et around the wrap body, traverse basal Shi pocket, the A quartet at 2010). microtubule nucleated al., the 2006; are as et (MtQ), known Zhou microtubules, 2005; four al., al., of et set unique He 2007; et al., et Selvapandiyan (Broadhead TbLRRP1 ae oehrteepoencmlxsrglt h position the regulate complexes protein these together Taken Trypanosoma 3173 Journal of Cell Science yoiei Hmatne l,20;d rfere ta. 2008; al., in et involved Graffenried is de and 2007; bodies al., basal evidence et the (Hammarton Other to cytokinesis localize 2008). can al., it of et that process suggests Graffenried the in (de implicated duplication earlier bilobe had we which (TbPLK), PLK 3174 niiino L1fnto ed odfcsi pnl pole 2006). spindle al., 1996; et in McInnes Nigg, 2006; defects and al., (Lane et to Peters congression leads chromosome 1995). function and al., formation PLK1 et Golsteyn of 1994; al., Inhibition et midbody (Golsteyn and cytokinesis spindle central to the prior to localizes nuclear finally the and of breakdown envelope, the following spindle kinetochores to the migrates 2005). and then al., poles centrosome, et the Preisinger on found 2003b; initially al., is et PLK1 Elia 2003a; al., as et such (Elia kinases Cdk1 upstream of action to the targeted by destinations be programmed to including a them contain allows PLKs that division, 2009). cytokinesis domain Glover, phosphopeptide-binding and cell and Archambault 2004; al., in congression et (Barr roles chromosome are separation, important kinases centriole plays of polo mammalian biogenesis PLK1 In the eukaryotes. The cells, higher regulate in elongating. to structures known cytoskeletal is many are that flagellum precise proteins at the migratory complexes new as of assembly times the to coordinate structure one to from migrate another to ability the is positioning flagellum n ftems motn etrsfrayptnilrgltrof regulator potential any for features important most the of One In .brucei T. ora fCl cec 2 (13) 125 Science Cell of Journal hr sol igePKhomolog, PLK single a only is there , .brucei T. Results defects. cellular irreparable duplication to cytoskeletal show leads key data events of Our concerted regulator segregation. in vital bodies, a events basal is the TbPLK of is that case duplication, it prevents the as kinase structure in the each or, of to depletion localizes that TbPLK and of that through duplicating migrates show assembly TbPLK We during how cell. extend explain the they could present which as is FAZ, microtubules new and the the initiates of it tip as the MtQ at new bilobe, resolution. the bodies, temporal with of high basal associates with the The established TbPLK events been to flagellum. has respect FAZ duplication and the with FPC kinase of with the associated of sequence migration structures ordered cytoskeletal the the explain mitosis. in can role PLKs, a mammalian play to to appear contrast not In does 2008). TbPLK Wang, and Umeyama oteblb n ial soitswt h rwn i fthe of tip growing the with associates finally progresses and then cell bilobe body, during the basal that the to showed with others colocalizes of TbPLK those division and of results series previous a Our of duplication of the organelles end with cytoskeletal anterior coincides the and to cell posterior the the from migrates TbPLK eew hwta h oeetadfntoigo TbPLK of functioning and movement the that show we Here .brucei T. n htitreigwt hs highly these with interfering that and n yoiei (VII). cytokinesis (VI) and karyokinesis by into followed (V), resolve structures kinetoplasts two bodies the basal segregate, the to As begin FAZ (IV). new extend the to while continues separate, replicated to newly begin The bilobes (III). duplicate bilobe the FAZ Subsequently, and (II). pocket own flagellum its new into then the FPC segregates and which pocket duplicate, The new (I). two bodies of probasal formation new the a by nucleate followed body. to flagellum, basal matures the body is probasal duplicate The to structure first the n ioe h lglu sahrdaantthe against FPC adhered the is by flagellum clinched The tightly bilobe. is and which of top pocket, flagellar the the from emerges flagellum. flagellum single The the and nucleates body which probasal body, the basal to near the cell the in of present posterior is kinetoplast the The with associated flagellum. structures cytoskeletal the of with associated the structures Cytoskeletal 1. Fig. elbd yteFZ ( FAZ. the by body cell .brucei T. flagellum. B uigtecl cycle, cell the During ) ( A ceai view schematic A ) Journal of Cell Science 2 IB1 hc ak h lgla oktcla;ad()the (3) and with collar; pocket determined flagellar (C4); cell the TbCentrin4 was marks marker during which bilobe refine kinase BILBO1, and body TbPLK (2) the basal To the of of of (1) to: phase. movements respect each localization duplication the on the of division, present their understanding is during our TbPLK 2006; organelles Wang, 2008). and Kumar these Wang, 2008; and al., et Umeyama Graffenried (de FAZ new el tie o h akr fteblb,bslbd,FCand localization FPC TbPLK body, of basal intermediates bilobe, of the images of Additional markers FAZ. the for stained cells line. temporal cell a this achieve for to minutes able 20 were of we resolution acquiring images, and By 4500 1976). (Sherwin Hall, analyzing in previously and and Barford pattern described 1959; Contois, localization been 1989; TbPLK has Gull, each as used of detail was duration temporal culture asynchronous the an estimate in of stage to frequency cycle relative cell used The each be 2003). at (McKean, to cells marker The it cycle flagellum. allowing cell new a nucleus, as the the of before basal duplicates emergence as kinetoplast the such localization cues and external TbPLK duplication of of body observation the order by temporal established The was FAZ1. marker FAZ i.2sostetmoa oaiainpten fTPKin TbPLK of patterns localization temporal the shows 2 Fig. o2.2 to oad h neiro h el(i.2 Ig.TPKsann was staining TbPLK IIIg). 2, (Fig. cell the of anterior bilobe, the towards the point this 1.3 At between normally IIc,IIIf). the and was of became 2, which side visible, anterior (Fig. FAZ the became to structures FPCs moved the duplicated labeling discrete TbPLK of the Two of end most IIe). then and posterior 2, 2, FPC (Fig. (Fig. the structure the apparent Ib). bilobe to the from 2, on Localization shape (Fig. lost IId,e). bar-like was shape a thin by form signal arch-like to formed identified appeared TbPLK an TbPLK structure, the form stage bilobe Subsequently, to this the staining, At contacted TbCentrin4 time. duplication that body this Basal projections the Ia). abutting 2, around FPC, (Fig. occurs stained structure and the body, BILBO1 of probasal the edge then and of exterior body kinase contour basal bilobe the small, The mature followed the the a S1b). both and Fig. to to body localized material localized basal (supplementary kinase the structure the between expression projection time in earliest tendril-like TbPLK available the At of are culture, TbPLK. express asynchronous point images, cells an of DIC In 60% S1–3. approximately with Figs along material supplementary marker, each with m bL n h lglu in flagellum the and TbPLK rlne ln t ao xsa bL migrated TbPLK as axis major its along longer or m muoloecneimages. and immunofluorescence schematics are the green in in represented shown events The cell cycle. the during events important duplication of timing the line shows black The respectively. e,h,j,k,l), red; (FAZ; red; anti-FAZ1 BB; b,d,g,i), + (Bilobe anti-C4 red; a,c,f), (FPC; anti-BILBO1 were with FAZ, labeled and FPC bilobe, and the a-l) green; (TbPLK; antibodies anti-TbPLK with kinase labeled The was (right). cells of in pattern TbPLK migration the of images immunofluorescence and various (left) at stages TbPLK of localization the and structures cytoskeletal associated flagellum- the of intermediates duplication showing schematics cycle. cell the during structures the cytoskeletal across duplicating migrates TbPLK 2. Fig. m n 1.8 and m .brucei T. m nlnt,grew length, in m eisof series A 3175 Journal of Cell Science oktrgo ftecl,wt h l n e t hw nturquoise. in shown MtQ new and old the with cell, the of region pocket band(lc o;c.TPKlbldtenwMQ(ht rohas,wihapasa hr rjcinnx otenwbslbd.()Amdlo the 5 n of NMtQ, (a) flagellum; model bars: new Scale A NFlag, MtQ. flagellum; (d) Flag, old body. (arrowheads). regio OMtQ, basal MtQ body; pocket new new basal flagellar the the old of the 1 to tip OBB, of (a) the next flagellum; of magnification bars: old projection magnification Scale higher OFlag, higher short turquoise. a A body; in a (b,c) and basal shown as A. MtQ new (b) in appears new NBB, selected as which and labeled antibod was old arrowheads), cell, anti-TbPLK the cycle The (white with with (a) cell stained MtQ cell, cycle. detergent, the new the with in of the extracted region early labeled were pocket cell cells TbPLK flagellar whole A c). labeling, (b,c). box; this EM (black of source by obtained the examination identify for To stained blue. in negatively DAPI and red, in labeling i.3 bL oaie oteMtQ. the to localizes TbPLK 3. Fig. (Lacomble body basal is new MtQ the new of the side cells, anterior by the anti- dividing visualized on In with then initiated this (EM). and labeled identify microscopy stained, clearly were electron more negatively cells To antibodies, initiation 3Aa). extracted the TbPLK (Fig. near projection, MtQ very is new tendrillar tendrillar that a the in body of present basal the At site was the cell. TbPLK to throughout cycle, the next cell throughout projection MtQ continues the the migrates of and with kinase stages TbPLK the early cycle of how association explain cell occurs The would FAZ. MtQ the the the of of in extension initiation The the early of 2009). part very al., becomes then et the and at (Lacomble FPC initiates FAZ the which MtQ, through the passes of nucleus body, track basal the the with disappeared coincides cell kinase then the S3i). Fig. the and material for (supplementary kinetoplast cytokinesis signal to the prior and The of FAZ just tip 2k,l). while growing bilobes the (Fig. precede FAZ the segregated to segregated new preceded continued TbPLK that the the IVi,j). 2, structure (Fig. from a structure duplication in migrated Bilobe IIIh). 2, concentrated bodies. (Fig. then FAZ basal new the of TbPLK of initiation pairs with both coincident appeared on visible still 3176 h irto fTPKfo h otro oteatro of anterior the to posterior the from TbPLK of migration The ora fCl cec 2 (13) 125 Science Cell of Journal ( A bL aesatnrla tutr al ntecl yl.TPKlbln ssoni re,bslbd n bilobe and body basal green, in shown is labeling TbPLK cycle. cell the in early structure tendrillar a labels TbPLK ) rqetylbldwt niTPKatbde (supplementary antibodies anti-TbPLK was flagellum structure new with This the 2004). labeled of al., et tip frequently (Briggs The the identifiable at EM. clearly shape is TbPLK by triangular that distinct examination whether a for has test stained a FC anti-TbPLK negatively with as To labeled and flagellum were 2001). cells antibodies old extracted FC, the al., the to using et localized by This new (Moreira-Leite the body S4A). position cell guide helps Fig. the which FC, material along the flagellum (supplementary by mediated flagella point is contact interaction two DIC the to of the corresponded inspection signal of Close this separation. most that body showed was the images of basal labeling side to This opposite prior the IIe,IIIh,IVi). to on intense 2, and appeared (Fig. FAZ that new membrane seen the plasma was of tip signal the punctate beyond small late be a and FAZ, new early the This at 3Ba–d). MtQ (Fig. cycle. the tip cell with the its of of associates on part stages TbPLK found using had become be that had seen MtQ could shows and that TbPLK new pocket FAZ, to flagellar the the similar the was Once MtQ beyond pattern new extended a 3Ab–d). the (Fig. cycle in cell immunofluorescence the TbPLK of with stages early labeled At 2010). al., et uigtepro hnTPKi rsn nteblb and bilobe the on present is TbPLK when period the During m ,()5 (b) m, m ,()50n,()50n.()Amdlo h flagellar the of model A (d) nm. 500 (c) nm, 500 (b) m, m ,()50n.( nm. 500 (c) m, B ela ae tg ftecell the of stage later a at cell A ) e,adthen and ies, wMtQ; ew was n Journal of Cell Science salse,w ogtt dniyteefc fdpeigthe depleting of been effect had the cycle identify cell to the during sought TbPLK we of established, migration the Once duplication body basal maturation affect or not does depletion TbPLK MtQ. the distinct with be associates to that appears one TbPLK of the pool from This S4B,C). Fig. material lglu per ik.Teshmtc ntelf ersn h ulcto ftebslbd n h lglu.Teudrcr niae h point the indicates a underscore assembled The are flagellum. bodies the probasal and new body then basal (e–g) the of matures duplication body 5 the probasal bar: represent the left Scale condition the control duplicate. on the structures schematics In The regions. (i–k). body appears basal flagellum the of magnifications are flagellum. insets new a ( nucleate induction. and duplicate RNAi mature, BB 4. Fig. place. unlikely taken it had making ingression TbPLK observed, furrow upon rarely incorrect that were However, cells (0N1K). 0N1K nucleus kinetoplast depletion, cell a the a arise produces only also cells can which controls containing cells furrow, TbPLK-depleted cleavage 2N1K mispositioned with (2N1K). the a from kinetoplast of compared the before 25% doubled duplicated In and two (2N2K) 4A). nuclei (Fig. and two kinetoplasts nucleus with cells one two one while and with decreased, (1N1K) cells (1N2K) was of kinetoplast kinetoplasts number content one the and RNAi, DNA of nucleus induction When hours RNAi RNAi 18 S5B). TbPLK after of of quantified Fig. hours hours 16 material within (supplementary depletion 8 arrest and TbPLK growth within S5A). to (de was control led Fig. occurred material depletion of cytokinesis (supplementary and TbPLK induction blotting undergo efficient that Western showed to extremely 2008). cells bilobe inability al., in doxycycline-treated and defects et an content, kinase Graffenried DNA and the system abnormal depletes with RNAi duplication cells effectively doxycycline-inducible in TbPLK a results Previous localizes. that against it shown directed which to has organelles work various the on kinase B oto el tie ihat-beti2atbd C;gen,at-BCatbde TB;rd n AI(N;bu) The blue). (DNA; DAPI and red) (TBBC; antibodies anti-TBBC green), (C2; antibody anti-TbCentrin2 with stained cells Control ) m m. ( A h ecnaeo el ihteidctdnmeso uliadkntpat fe 8husof hours 18 after kinetoplasts and nuclei of numbers indicated the with cells of percentage The ) perneo h rbslbde a emntrdwt an basal the with of monitored component a be is can which After (C2), bodies TbCentrin2 2008). probasal to The antibody 1989). al., the Gull, of and et bodies, (Sherwin probasal appearance cycle Absalon new duplication assemble basal the 1999; against bodies completing mature basal al., two with antibodies the selectively et maturation, coiled with associate (Dilbeck a to protein, bodies tracked shown (TBBC) protein 2010). be component al., coil body et can basal (Lacomble trypanosome flagellum the maturation gains new which a This body, nucleate probasal the to of capacity maturation is process and this a duplication hypothesis, maturation, this body conducted. test basal was To separation that of kinetoplasts. possible accounting is segregated careful explain it of could kDNA, separation lack and/or the the duplication segregating body duplicated basal in the in the body defects importance basal that of the the Considering segregation suggests 2007). of al., from et which cells (Hammarton stems structures 2N1K depletion, in defect kinetoplast TbPLK kinetoplast kinetoplast single the upon the that of increases shown content had (kDNA) work of DNA replication Previous proper the kinetoplast. for necessary the is kinase the that suggested salse,te ei osprt,wihdie h eaainof separation the been drives have which bodies separate, new basal to the new begin of the they in established, assembly Once assembly monitored. and be position its present to the also in flagellum allowing is early flagellum, C2 the 2007). structure al., in the et (Kleylein-Sohn to cells recruited mammalian is and body aa oyrpiainocr n11 el.Tefrtsaeof stage first The cells. 1N1K in occurs replication body Basal samples TbPLK-depleted in cells 2N1K the of appearance The bL n h lglu in flagellum the and TbPLK .brucei T. twihthe which at h new the s 3177 Journal of Cell Science ubro aueBsadtenme fnce,adtenme ffael n h ubro ulii igeclsi bL-eltdadcnrlcells. control and TbPLK-depleted in cells single in nuclei of number the and flagella Scal of cells (m–o). number TbPLK-depleted flagella the of four and subset and nuclei, A bodies of basal e–g). number b eight (a–c, the the subsequently occur and of and still duplication BBs (i–k) duplication the mature BBs BB represent of TBBC-positive and left number four maturation the generating body on maturation, probasal schematics BB TbPLK, The 5 of of blue). round absence (DNA; second the DAPI a TbPLK. In and underwent of flagellum. red) independent the (TBBC; are flagellum and antibodies the anti-TBBC body of green), nucleation and (C2; duplication antibody BB TbCentrin2 maturation, body Probasal 5. Fig. body basal total of ratio The the 5B). in (Fig. changed was cells maturation or TbPLK-depleted number body basal whether test that of suggesting independent flagella, is structures and these TbPLK. of bodies duplication 5Am–o). capable basal and (Fig. still maturation new were flagella but generating four cytokinesis undergone of with not had along cells These positive, which of TBBC four Other structures, occur. were C2-positive second not visible 2N should a eight which contained many undergone 5Ai–k), cells the have In (Fig. to of maturation 5Ae–g). of appeared (Fig. placement round bodies cells probasal close 2N the the the in cells, of bodies and detachment basal flagellum 5Ad,h,l) the duplicated new (Fig. were a flagellum defects nucleating clearest new of The capable 5Aa–c). was (Fig. newly body the TBBC and basal appeared became mature bodies body probasal probasal C2-positive The the positive, defects. any without proceed visible, separate begins are to begin As flagellum bodies spots basal 4Bi–k). 4Bm–o). of (Fig. new (Fig. pairs C2 bodies the probasal proceeds, the four the division cell of of assembly afterwards, the formation indicating Soon the TBBC the 4Be–g). At becomes (Fig. body 4Ba–c). and (Fig. probasal and the flagellum mature positive duplication the the of both stages with labels earliest along C2 labels body, 1N2K whereas TBBC the body, probasal division, to from basal emerged progress mature just to the have that cell cells the In leading state. kDNA, replicated the 3178 m ofrhrcnimti eut ecluae eea aisto ratios several calculated we result, this confirm further To to seemed duplication body basal cells, TbPLK-depleted In .Teudrcrsidct h on twihtesrcue nraei ubr ( number. in increase structures the which at point the indicate underscores The m. ora fCl cec 2 (13) 125 Science Cell of Journal h bL-eltdsml sdet h lglu biogenesis cytokinesis. flagellum of in absence the cells the 2N to in of due continuing not subset is process a flagellum. is sample in the TbPLK-depleted TbPLK flagella or the body additional that body basal of the suggests appearance basal of The This duplication TbPLK- in the in cells. for indistinguishable necessary defects control were and any ratios depleted three identify to All number would TBBC- maturation. flagellar of of number ratios and the number nuclear in nuclear changes to cause structures Similarly, would positive increase. duplication to early ratio in or the duplication delay whereas decrease, of a to basal-body– rounds or ratio the multiple Defects two cause would nucleus. have duplication single body cells basal a these and because pairs the four cells, probasal-body basal-body– that 1N2K be all two cells should in 1N1K and or ratio duplication In body 1N1K) (2N2K). basal nuclei undergone (early have two nucleus and probasal single pairs and probasal-body body a basal a and and have cells cells body 1N1K these early because in cells two 2N2K be all should number nuclear to number ulcto rmtrto,coe npcino h data the be of might body structures inspection basal duplicated in the closer role of a separation maturation, that play suggested to or seem not defect duplication does segregation body TbPLK basal Although a to leads depletion TbPLK oto el.Tedsac ewe h aa oypiswas pairs body on basal conducted the between was and distance EM TbPLK-depleted The of To cells. stain 5Ae–l). control cytoskeletons negative (Fig. whole-mount together had effect, close extracted samples were this TbPLK-depleted that in bodies quantify cells basal 2N of the pairs of Many failing. 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FPC (Fig. cells, nuclei. control of In number extracted TbPLK. the the of of states terms in DNA in the only analysis, categorized 1N1K poor the were simplify between cells To distinguish is cells. to 1N2K component difficult and FPC preservation it extracted the making this, against cytoskeletons, Kinetoplast test antibody an To control with BILBO1. FC. and stained the TbPLK-depleted and by of from cells because prepared flagella pocket were the same cytoskeletons the of could from linkage FPC its emerging the the flagella during duplicating two region in to this lead Difficulties in and 2a). localize cytoskeleton, (Fig. the partially to duplication to it found links was and TbPLK pocket the demarcates FPC ttelgtmcocp ee htapae oso h new the show old. to the as appeared position which same that the FPC, level from single emerging microscope images flagellum a our light with from consistent the is emerged at This 7Cd,e). flagella (Fig. this sample, new enlarged emerges is TbPLK-depleted seemed it and test the time In flagellum old the 7Ca–c). to by (Fig. the new body EM FPC cell and the the stain pocket from own conditions negative its within normal to confined subjected Under then hypothesis. control and and flagella. TbPLK-depleted two cells accommodate from to extracted FPC form were old to the failure Cytoskeletons force The could 7Aj). FPC (Fig. new flagellum a old the as position same nteTPKdpee ape ay2 el ihasingle a with cells 2N many sample, TbPLK-depleted the In of depleted cells in impeded is FPC new a of formation The bL n h lglu in flagellum the and TbPLK m m. C ersnaieiae fcnrladTbPLK-depleted and control of images Representative ) A , B h ai ftelnt ftenwto new the of length the of ratio The ) .brucei T. ,adclsthat cells and ), 3179 Journal of Cell Science emdt mrefo h aepit() e anfe iwo h oe eini hwn httefael eeecrldb igeFC(arrowheads FPC single a by encircled were 5 flagella (a) the bars: fla that Scale had body. showing frequently basal d stage old in cycle OBB, region cell flagellum; boxed similar old the a OFlag, of at body; view cells basal Magnified sample, new (e) TbPLK-depleted treatme NBB, (d). each the flagellum; in point In new state same b,c). nuclear NFlag, the (arrowhead; Each from FPCs 2N). emerge separate or to two (1N seemed had nuclei of (a) number flagella the emergent to two according categorized are cells ( The 100%. cells. TbPLK-depleted in defect duplication vdn Fg A–,arwed) hs eet will defects 8Ap–t, (Fig. that images DIC cells in These were seen Numerous arrows). were FAZ detail. flagella new new more arrowheads). detached in the had described of doxycycline- be 8Ak–o, formation the (Fig. subsequently the In filament. in evident 8Aa–e, FAZ defects and the (Fig. their to cells, cells attached stages to treated 1N1K were adjacent flagella 2N2K of body new and and subset cell old a 1N2K the Both in the arrowheads). emerging during FAZ elongating new the (Kohl that with DNA 2008). antibody detect al., to an et that DAPI Vaughan with and 1999; likely al., FAZ1 cells we et protein seemed the this, FAZ test stained the it To defect. detects and FAZ flagellum, a TbPLK to new lead depleted for might essential the kinase is the which of depleting FAZ, new adherence is the of TbPLK of the tip that growing Considering numbers the flagella. at large new present detached contained had samples that cells assembly TbPLK-depleted FAZ our new in All defects causes depletion TbPLK 5 bars: Scale (j). seen are state 2N the in FPC 1 with cells depletion, TbPLK Upon stage. 1N the at occurs (b) duplication. duplication FPC ( in blue). defects (DNA; causes DAPI depletion TbPLK 7. Fig. 3180 oto el hwdnra A opooyadnumber, and morphology FAZ normal showed cells Control C hl-on yokltn fTPKdpee n oto el eengtvl tie o iulzto yE.I h oto ape elwith cell a sample, control the In EM. by visualization for stained negatively were cells control and TbPLK-depleted of cytoskeletons Whole-mount ) ora fCl cec 2 (13) 125 Science Cell of Journal A P ulcto tgsi eaint A ulcto.TenwFC r niae yarwed.I h oto odto,FPC condition, control the In arrowheads. by indicated are FPCs new The duplication. FAZ to relation in stages duplication FPC ) bL-eltdadcnrlclswr aee ihat-IB1atbd FC re)and green) (FPC; antibody anti-BILBO1 with labeled were cells control and TbPLK-depleted A 4% i.8) ewr nbet sals rtrato criteria establish to single unable a were or (50%) We with snFAZ 8B). defects, an Fig. FAZ either (42%; The containing strongest 8B). 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Journal of Cell Science eltdcniinaeshown. are condition depleted ( arrowhea 100%. (k–o, to FAZs new the 5 cells, bars: TbPLK-depleted Scale In observed. (c–e). thereafter were elongated arrows) and (p–t, (b), flagella stage 1N1K detached the and in defective, initiated frequently is FAZ new The FAZs. duplicating A eet vni P ulcto rcee omly These normally. proceeded duplication cause FPC cells could if these depletion If even TbPLK snFAZ. defects that an 2N FAZ suggest or would of FAZ it single examples present, a were for and FPCs looked two then with cells we and and BILBO1 for simultaneously, samples depletion. FAZ1 TbPLK-depleted TbPLK FAZ stained upon we defects this, affect TbPLK test FAZ To to If only also possible have be 2008). should that can it al., cells FAZ, observe new et FPC a Bonhivers of assembly new 2008; the affects whereas directly al., a FAZ, et new of (Shi the a biogenesis formation of of depletion formation the and the upstream blocking linked blocks the C2 are TbPLK- in protein FAZ is the defects bilobe and which by bilobe of FAZ, influenced The aspects The be structures. indirect. some could the last, then be assembled exist, might does phenotype them. depletion among hierarchy hierarchy a a reflect may If which had order, flagella prescribed a detached in 8D). with (Fig. Cells than formation 8C). 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Fig. h yokltleeet htTPKitrcswt duplicate with interacts TbPLK that elements cytoskeletal The B A hntpsosre ncnrlclsadTPKdpee el tdfeetpit ftecl yl.Teaern NKDAsaei only is state DNA 2N1K aberrant The cycle. cell the of points different at cells TbPLK-depleted and cells control in observed phenotypes FAZ ) D ecnaeo Nclswt bomlFZ ete A rsFZ rnra Asta aedtce e lgla nydt o h TbPLK- the for data Only flagella. new detached have that FAZs normal or snFAZ) or FAZ 1 (either FAZs abnormal with cells 2N of Percentage ) C bL elto ed odtcmn ftenwfaelm nbt n ,ec odto nec elccesaesums stage cycle cell each in condition each C, and B both In flagellum. new the of detachment to leads depletion TbPLK ) bL-eltdadcnrlclswr tie ihat-A1(A;rd and red) (FAZ; anti-FAZ1 with stained were cells control and TbPLK-depleted eebe h rc fteMQ h iaei rsn nthe on present is kinase The MtQ. the of track the resembles pocket. ordered an own of its highly within inheritance resides the the that for flagellum necessary functional prohibits attached, events proceed structures cytoskeletal of these series karyokinesis ordered of of the production Perturbing duplication flagella. the damaging and to detached to with fail leading cells especially multinucleate cells, duplication FAZ TbPLK-depleted are and in FPC normally flagellar defects the These and because segregate, was properly. the to structures: TbPLK duplicate these fail theory, in bodies defects this caused basal which test RNAi, of To by duplicate, inheritance depleted elements. or structures replication cytoskeletal these the pattern these which triggers This FAZ. TbPLK in new that order the suggesting of the tip matches growing the closely bilobe, to FPC, to finally, the MtQ, and, to the function progresses FC to subsequently bodies, localizes that basal initially the kinase structures then The cytoskeletal flagellum. the important position depletion its several TbPLK of of effect the movement on determined the and detail cycle in cell the charted throughout have we article, 2N this In the of 18% Discussion constituted defects S6). FAZ Fig. material with (supplementary population cells FPC two A A lnaindrn h elccei oto el.Arwed indicate Arrowheads cells. control in cycle cell the during elongation FAZ ) h irto fTPKdrn h elcceclosely cycle cell the during TbPLK of migration The m .Tetr nA eest e A n2 el hti ne 5 under is that cells 2N in FAZ new to refers snFAZ term The m. bL n h lglu in flagellum the and TbPLK .brucei T. s were ds) 3181 m min Journal of Cell Science nofu iceetps edi-iesrcueta ak the marks that structure tendril-like is a It types: TbPLK FPC. of discrete the localization four pocket and flagellar into structure the categorize bilobe to the possible with coincides partially to defect. segregation necessary product body the basal are are TbPLK-depleted they a in that flagellum of cells suggesting flagella, 2N1K and two the contain all kinetoplast samples Almost zoid. the the the because of produce flagellum from in one only arise copy cells have that also zoid one should of cells zoid a number 2N1K of limited production a samples. a as TbPLK-depleted show known kinetoplast, counts our This a DNA 2008). only the Our al., containing et zoid. of cell Shi a segregation 1999; generate al., or would et if karyokinesis arise (Ploubidou also nuclei to could duplicated prior cells that 2N1K occurs kinetoplast kDNA. single the cytokinesis a of not copies leaves do two separation pairs contains of body bodies basal lack probasal pairs formed This new newly body separate. two the probasal of basal but formation normally, cells, the proceed the TbPLK-depleted of and In maturation separation 1991). body the Gull, and to structures (Robinson coupled discrete two directly depletion into The kinetoplast TbPLK is 2007). duplicated al., by the et of (Hammarton generated division kDNA of cells equivalents that 2N1K two shown contain effectively has in to work kinetoplast failed Previous the have DNA. that kinetoplast cells their segregate 2N2K are samples depleted upstream 2004; al., the et in (Barr 2009). TbPLK change Glover, for and a sites Archambault reflect binding towards generate may flagellum. that function the kinases specialized of in positioning proper the change become the as This assure such that for has structures FAZ case and cytoskeletal FPC the TbPLK of TbPLK not duplication is PLK1. the However, controlling which and mitosis, equivalents. for cdc5 dispensible centriolar function be this to its conserves their (Snead appears TbPLK spindle 1993). partitioning replicate al., appropriate et an of but Kitada can form 2007; bodies, to al., pole cdc5 apart et spindle them homolog the 2009). move unaffected al., the as cannot PLK et known Tsou of is equivalents, the 1996; centrosome Nigg, positioning duplication the lacking and Lane the centrosome in Yeast 2006; al., and defects while et (Hanisch centrosomes causes poles, the cells the yeast. of spindle mammalian budding of function in in the separation and depletion with cells mammalian consistent duplicate. PLK1 in duplication is they kinase body basal as which homologous either them maturation, on effect with or no has associated depletion remains TbPLK and by bodies TbPLK of migration kinesins. the positive-end-directed 2009). the al., with facilitate underlies et association that Lacomble could that the 1995; microtubules arrangement al., array the et at This (Robinson subpellicular to cell body the the opposed of of basal surface as rest the cell, the the from constitute of of ends nucleate end positive The microtubules posterior 2003). al., MtQ et the Neef the of 1999; to migration al., shown the et been PLK1 facilitate (Feng has kinase could cell. and which the microtubules kinesins, throughout of with subset interact the move a with to to localizes, interacts extension directly TbPLK binds selectively its kinase which uses the and to MtQ that structures possible tip the its it comes all on MtQ making The is with FAZ. and the contact body, of basal into part the becomes to microtubules next the assembles once first it as MtQ 3182 bL hnmvst h lgla oktrgo,which region, pocket flagellar the to moves then TbPLK TbPLK- in present are that cells 2N1K aberrant the of Many basal the to localizes TbPLK cycle, cell the in phases early At ora fCl cec 2 (13) 125 Science Cell of Journal asg ftenwfaelmtruhteodFCmgtbe might intermediate FC. FPC the The some through old flagellum flagella. in old the the through to caught both attachment flagellum its is accommodate new by facilitated the FPC can of the the that passage through that cells, thread state some to or duplication in able new is FPC FPC and flagellum same old old new the the The through that pocket. exit suggesting old length to the normal appear of with flagellum be associated to formation appears remains FPC flagellum new and new depletion the TbPLK but of (Bonhivers blocked, case pocket is the flagellar new the In a 2008). to lacks a al., led that et this flagellum of but a BILBO1, of of segregation duplication formation depletion FPC upon and of observed Failure been assembly 2008). has al., et the The Graffenried (de in structure FPC. defects new causing the allow tested. on would be proteins to BILBO1 hypothesis FPC-resident this of additional depart of deposition also identification might kinase the The structure recruitment. present before is arch-like BILBO1 TbPLK new The that to suggesting FAZ. prior FPC, new nascent a the represent of might that emergence structure punctate the a the marks and traverses protrusions bilobes, that with segregating structure and bar-like structure a duplicating arch-like bodies, basal an the MtQ, contact that new a of initiation h cuuaino NKclsadtefc htte21 cells 2N1K the that at by fact the shown fail and is to cells This 2N2K appear prematurely. of they it accumulation the defects, trigger TbPLK- than FAZ Although rather 2008). major cytokinesis Gull, 2010; have and (Vaughan, cells Vaughan cells depleted 2003; daughter al., the et of Kohl FAZ cause in partitioning can defects which unequal and furrows, the FAZ, misplaced cytokinesis new to the lead completing to by frequently seems dictated formation furrow for position cleavage a the at essential of occur Ingression 2006). also al., et is (Ralston motility. proper motility lack will division the Proper cell of a subsequent one of any that ensures of production and progeny The reversed flagella. be cannot detached flagellum proper to detached leads the which assure FAZ, to new is TbPLK of flagellum. the function the of organelles main reinforces inheritance other the This the TbPLK. that of by idea function controlled the the are of inheritance to orientation whose similar and is position role the flagellum proposed templating new the in precise unknown, is structure the FC this Although the of on identified. plays be kinase the the to is that FC new TbPLK role the stages. the later of as at component visible diminishes first still to signal is but prior This extends, 2001). flagellum just al., until et FAZ. present (Moreira-Leite altogether. new cell is the of the FAZ from tip of disappears the growing it end on when the the cytokinesis, signal on with TbPLK present overlaps The is which TbPLK structure, the are visible, this Once FAZ is structure. and cytoskeletal FAZ bilobe continuous the new larger that tip a suggests the of This at segments FAZ. reside to growing appears the duplicated TbPLK of the point, of this structure tip At the structure. anterior departing bilobe the Before at FAZ. concentrates new TbPLK the be entirely, of to tip and remains bilobe the this although BILBO1, it of as directly. absence properly, tested segregate the that or suggests in duplicate depletion to does TbPLK fails upon pocket FPC flagellar new the a of absence The bL a rvosybe mlctdi ioeduplication, bilobe in implicated been previously has TbPLK bL elto asssrn eet nteetnino a of extension the in defects strong causes depletion TbPLK FC the to localizes also TbPLK cycle cell the of point this At to migrates TbPLK complete, is FPC the of duplication Once Journal of Cell Science ih3 S.Tegiswr hnwse nPS ie n25 glutaraldehyde 2.5% in fixed PBS, nm aurothioglucose. PBS in 10 0.35% washed in with by then stained 1:10 followed were negatively diluted grids PBS), and (BBInternational) The in BSA. antibodies BSA 3% anti-rabbit with 3% goat in gold (1:50 colloidal antibodies anti-PLK polyclonal tie ihscnayatbde.Teclswr ahdi B,mutdin described mounted previously PBS, as in visualized 2009). washed and al., Biotech), were et (Southern (Morriswood cells then G The PBS, Fluormount in antibodies. anti- washed DAPI 1:800) secondary 1:200, anti-TBBC with anti-FAZ1 or 1:30 1:50, stained anti-C2 diluted 1:80, anti-C4 (anti-TbPLK 1:8, antibody BILBO1 primary with stained pcfcodr bL speeto aho hs tutrsas structures these of each on present is a TbPLK in bilobe, partitioned order. and body, assembled specific be basal therefore must the FAZ as and FC such FPC, in properties, flagellar division these cell control of part essential production the from arise not zoids. do of depletion TbPLK by produced nlzd o M 510clswr nlzd nalhsorm,tbe n the and tables histograms, all In analyzed. were were cells condition 85–120 each for EM, cells For 500–550 analyzed. immunofluorescence, of quantification For analysis Mathematical mM 1 6.9, pH PIPES M 0.1 NP40, (0.25% grids extracted nickel detergent MgCl carbon-coated and and formvar- mesh) glow-discharged, (200 onto settled were Cells microscopy electron Immuno-gold 1 at resuspended and washed were Cells microscopy Electron at methanol in fixed and coverslips to adhered were Cells Immunofluorescence IgG mouse of Robinson anti-C2 (University Derrick Pays and Etienne from Anti-C4 from anti-BILBO1 Belgium). anti-TBBC UK), France), Brussels, Oxford, Bordeaux, from of of (L3B2) (University anti-FAZ1 (University sources: following Gull the Keith from obtained were antibodies The 2008). al., Antibodies et Graffenried integrated (de stably described tetracycline-inducible, previously al., as a et cultured Graffenried with were (de previously Cells (Wirtz line described 2008). University) been cell has Rockefeller TbPLK (The 29.13 against construct Cross The RNAi G. 1999). by al., supplied was et line cell 29.13 The culture Cell Methods and Materials cytoskeleton. trypanosome for the configured of been features has the unique therefore licenses, the and it cycle, duplication cell whose the organisms,organelles during other pattern in localization shares homologs its its TbPLK with but kinases. mobility the of for degree the sites high this binding by concert destroy in times and act create specific which phosphatases, to at and kinases localize their upstream of to in action PLK failure programmed flagella. a misplaced are and causes homologs detached kinase to leads the which inheritance, of depletion duplicate; they ocnrtoso P4 02%a 0 at (0.25% NP-40 of concentrations B ro ofxto.Teclswr eyrtdi B n lce vrih at overnight blocked and PBS in rehydrated in were washed cells and The anti-C2) 4 with fixation. anti-TBBC to for prior 1% PBS anti-FAZ1, with anti-BILBO1 for niTPKhsbe ecie rvosy(eGafnide l,2008). al., et Rabbit Graffenried S7. (de Fig. previously material The described supplementary respectively. been in TbCentrin2, shown has is anti-TbPLK and antibodies TbCentrin4 these of recombinant specificity against raised were o ulcto sn htso S otae(db ytm,SnJs,CA). Jose, was San Systems, quantification (Adobe software Image CS4 Photoshop Germany). prepared using were figures publication System and for FIVE (http://rsbweb.nih.gov/ij/), analySIS 1.42q Imaging ImageJ and with (Olympus) (Soft conducted camera digital 0.5% microscope iTEM Morada electron with transmission a software, KeV Japan), stained 80 2.5% Tokyo, TEM and in Ltd. 1210 (Joel fixed JEM water, (Ho JOEL then in a and seconds with twice temperature 15 visualized room for rinsed at (Sigma-Aldrich) buffer, min aurothioglucose 3 PEME for in extracted PEME detergent glutaraldehyde in were NP-40 cells 1% The with grids. copper formvar-coated glow-discharged nuae o iue tro eprtr ro e c nPM ufr( mM (2 buffer PEME were in ice cells wet case, on MgSO latter or mM temperature the 1 room In at EGTA, cytoskeletons. minutes extracted 5 for detergent incubated for minutes 20 or ˚ ih3 S nPS xetfrat-IB1(oBA.Teclswere cells The BSA). (no anti-BILBO1 for except PBS, in BSA 3% with C neiac fa tahdadpstoe lglu san is flagellum positioned and attached an of Inheritance 2 .Te eete lce n3 S nPSadlbldwt rabbit with labeled and PBS in BSA 3% in blocked then were They ). 4 . MET,01MPPSp .)cnann different containing 6.9) pH PIPES M 0.1 EDTA, mM 0.1 , ˚ o niTPKwt niBLO,0.5% anti-BILBO1, with anti-TbPLK for C 6 .brucei T. 10 6 e li B,te one on mounted then PBS, in ml per ¨o ge l,21) el were Cells 2010). al., et ¨g h tutrsthat structures The . 1 oolnlantibodies monoclonal 2 20 ˚ o 5minutes, 15 for C ohvr,M,Nwci . aden .adRbno,D R. D. Robinson, and N. Landrein, S., Nowacki, M., Bonhivers, Sillje A., F. Barr, eg . og,D . amei . hs,D . og,D .adFri,D K. D. Ferris, and L. D. Longo, L., D. Chase, G., Palmieri, R., D. Hodge, Y., Feng, E. D. Shaw, N., Contois, Portman, R., S. Hart, S., Griffiths, H., Farr, R., H. Dawe, R., Broadhead, S. Vaughan, J., Davidge, F., Moreira-Leite, A., Baines, G., P. McKean, J., L. Briggs, J. R. Hall, and P. M. J. Barford, D. Glover, and V. Archambault, tg a acltdacrigt h equation: the to according calculated was as stage or bars When error as represented. either are represented is experiments values. (s.d.) independent numerical deviation standard three sample of the averages shown, the line, time nslr . fh,T,Hrigas . ohr,M,Weete,G,Heddergott, K., G., Wiegertjes, Hoepker, M., J., Boshart, S., F. Herminghaus, Ivins, T., Pfohl, W., M., J. Engstler, Chao, F., L. Haire, P., Rellos, H., E. B. A. M. Yaffe, Elia, and C. L. Cantley, H., E. A. Elia, E. Pays, and H. Alexandre, A., Cauwenberge, Van M., Berberof, G. V., Warren, Dilbeck, and H. H. Ho, L., C. Graffenried, de dieo muoE.W ol lolk otakalthe all thank to like also for Kotisch would reagents. the essential Harald of We contributed and reading that immuno-EM. laboratories critical Gadelha on for Catarina laboratory advice thank Warren We and the F. comments manuscript. of Max critical and Biology, members support Molecular the for for Austria) (Center Laboratories, Warren Perutz Graham thank We Acknowledgements bao,S,Binc,T,Bnies . ol . ae,N,Tuias . Buisson, G., Toutirais, N., Cayet, L., Kohl, M., Bonhivers, T., Blisnick, S., Absalon, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.101162/-/DC1 online Cell available material Supplementary of Warren]. Mechanisms [grant Graham to Molecular Fund W1220-B09 program Science number [grant Austrian doctoral Signaling Austrian and the Fund C.L.d.G.]; and by Science Warren Graham supported to P21550-B12 was number work This Funding siae rmtecluedniy(oti,1959). (Contois, density culture the from estimated to where sterltv rwhrt SewnadGl,18) h uaino ahstage each of duration The 1989). Gull, ( and subtraction (Sherwin by rate obtained was growth relative the is where h siaino h uuaietime cumulative the of estimation The 19) soito fpl-iekns ihapa,bt-adgmatbln na in gamma-tubulins and beta- alpha-, with kinase complex. stable polo-like of Association (1999). trypanosome. bloodstream the al. of et G. viability P. the McKean, for J., S. required Gaskell, is L., M. Ginger, K., M. K. Gull, and mediated. cytoskeleton is e105. organelle endo-exocytotic trypanosome the division. cell of orchestration cerevisiae. Saccharomyces of cultures asynchronous ufc ftrypanosomes. of surface P. Overath, and by N. Plks B. of M. regulation Yaffe, and and targeting domain. J. Polo-box substrate S. the phosphodependent Smerdon, for C., basis L. molecular Cantley, D., Mohammad, cultures. continuous of rate growth specific and density junction. transmembrane mobile regulation. and functions their in divergence Sci. Cell Trbnigdmi oaiigPk omttcsubstrates. mitotic to Plk1 localizing of domain body pThr-binding basal the in present protein coil brucei coiled a of brucei. Characterization Trypanosoma in biogenesis bilobe and Golgi for in pocket flagellar the of function and P. orientation Bastin, structure, and D. Robinson, J., x n ). t y m 1 stecmltv ecnaeo el nldn h tg nqeto,and question, in stage the including cells of percentage cumulative the is . satm on ae than later point time a is a bandfrom: obtained was .Cl Sci. Cell J. bL n h lglu in flagellum the and TbPLK 121 3704-3716. , 15) ieiso atra rwh eainhpbtenpopulation between relationship growth: bacterial of Kinetics (1959). 20) h lglacnetrof connector flagella The (2004). ,H .W n ig .A. E. Nigg, and W. 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