Ato o orsodne([email protected]) correspondence for Brazil. *Author 20261-232, Janeiro de Rio (Inmetro), Tecnologia e Qualidade Metrologia, Brazil. 21941-902, Janeiro de Rio Janeiro, aoiaL Alcantara L. Carolina cytostome-cytopharynx of the complex of structure three-dimensional The ARTICLE RESEARCH ß eevd2 a 03 cetd7Fbur 2014 February 7 Accepted 2013; May 20 Received 3 1 as such these family, of the intracellular from pathways parasites protozoan different most endocytic In the organisms. the through characterize manner, that and where orderly the compartments surface cell an of yeast, the in invaginations over and all passes, through distributed are mammals cell that membrane the in plasma enters characterized cargo well extracellular This is particles. and process molecules extracellular culminates of that internalization events the of in series complex a comprises Endocytosis INTRODUCTION tomography Endocytosis, Electron cruzi, Trypanosoma Cytostome, division. WORDS: cell KEY for preparation a the associated is and likely cytopharynx activity postnuclear is the endocytic the that Therefore, with remodeling reach path. undergoing its not structure, in did bend dynamic it a although to owing cycle, region cell G2 the the during of longer slightly phase was cytopharynx, cytopharynx the The of clusters. lumen forming the associated. along be distributed to unevenly was a found Cargo left were The and vesicles cytopharynx. ‘gutter’ where a side, the formed and microtubule-free cytopharynx reaching the membrane before accompanying sets ridge flagellar-pocket two cytostome preoral quartet triplet the a the The a and underneath followed – membrane, endocytosis. originated sets cytostome the that microtubule of underneath two started of process that three-dimensional composed produced the was the we cytoskeleton of including tracers, had complex, snapshots gold-labeled that cells up entire on Focusing taken vesicles. the electron and cytoskeleton reconstruct scanning surrounding focused-ion-beam to serial used and microscopy We structure. tomography remarkable electron this of morphology detailed endocytosis of site of main the is complex cytostome-cytopharynx The ABSTRACT enlgae ilgaEtuua iiaes i eJnio29192 Brazil. 21941-902, Janeiro de Rio Bioimagens, e Estrutural Biologia em Tecnologia otefc httefaelrpce ebaei o soitdwith associated not is due membrane is pocket endocytosis flagellar of the that site fact the emerges the in to flagellum restriction This the flagellar body. which cell at the the from site plasma to the the surrounds of restricted that invagination membrane an and 1993), Russell, and polarized (Webster pocket highly is endocytosis ieoi eMtooi piaaa Aplicada Metrologia de Diretoria Biofı de Instituto 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,22–27doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. rpnsm cruzi Trypanosoma ´ iaCro hgsFlo nvriaeFdrld i de Rio do Federal Universidade Filho, Chagas Carlos sica rpnsm brucei Trypanosoma pmsioe.Ltl skonaotthe about known is Little epimastigotes. rpnsm cruzi Trypanosoma 1,2 ` Cie s uin .Vidal C. Juliana , ˆ ca aVd,IsiuoNcoa de Nacional Instituto Vida, da ncias 2 nttt ainld Cie de Nacional Instituto , and Trypanosomatidae Leishmania 1,2 adre eSouza de Wanderley , ˆ cae ncia sp., epimastigotes ttsia nlss hc eur Drcntuto fa of reconstruction for 3D inappropriate is require and which the intensive as analyses, labor such electron very statistical serial invagination by is even or curved tomography sections ultrathin and serial by deep cytopharynx a cytopharynx the Reconstructing of length and Shape RESULTS arioe l,20) ahrta h lgla okt The pocket. flagellar the than of invagination rather deep a of 2000), consists (Porto- complex al., cytostome-cytopharynx complex main low-density cytostome-cytopharynx the et cycle, the and life Carreiro is the endocytosis of peroxidase of stage this site in albumin, de Nevertheless, and (Soares 1991). milieu transferrin, Souza, extracellular the from as – particles lipoprotein such endocytic macromolecules of intense types have different – endocytose and to vector able being insect activity, the of in Epimastigotes forms disease. proliferative Chagas of agent 1970), (Angelopoulos, cell the traffic. vesicular in preventing the elsewhere beneath membrane just positioned plasma are that microtubules subpellicular the o hssrcueprom h noyi ucinin function endocytic into the insights performs new epimastigotes. structure the provides this elements of associated how domains its cytostome-cytopharynx entire different and the complex of the model detailed of macromolecules. resulting of The function internalization the the in cytostome-cytopharynx define was in to tracers, This labeled of order endocytosis reconstruction. the of ion-beam-scanning visualization (3D) the to three-dimensional coupled focused tomography, by electron serial using followed and (FIB-SEM) by microscopy electron complex vesicles microtubule aligned cytopharynx the the of but function and unknown. an 1969), still origin are Also, Deane, the 2005). and and arrangement al., the (Milder with et aligned are cytopharynx VataruNakamura vesicles and (Martı microtubules 1989; of ridge’ set uncharacterized al., ‘preoral et the Pimenta as and glycocalyx known prominent a flagellar by is the characterized is and domain special cytostome This a the pocket. is between there that domain structural is membrane known endocytosis plasma the is how What understand structure. about to this through difficult known occurs it shortage make is The details structural complex. infective of little cytostome-cytopharynx the the process, of to organization endocytic epimastigote the importance its the for Despite disappears. complex of this form differentiation 1973). trypomastigote Souza, de the and (Meyer During is demonstrated been endocytic structure its rarely This although has the forms, capacity far. amastigote between so intracellular limit in described present defined is also cytopharynx no surface the is and cell There cytostome invagination ‘cytopharynx’. the the the forms at that called tube opening long is the the and ‘cytostome’ where the named membrane, plasma the hsstaini ifrn in different is situation This nti ok eeuiaeteutatutr ftecytostome- the of ultrastructure the elucidate we work, this In 1,2,3 n acs .Cunha-e-Silva L. Narcisa and rpnsm cruzi Trypanosoma ´ e-aooe l,1976; al., et nez-Palomo .cruzi T. h etiological the , 1,2, r h main the are * .cruzi T. 2227

Journal of Cell Science h yohrn fG aaie i o ec h otro fthe bend, of this posterior of the result reach not a did As cell. the parasites anterior 2E–G). G2 towards the (Fig. of cytopharynx cytopharynx cell to the the the close turned of region that region a the anterior nucleus, at bend reaching the cytopharynx, a was of and the there portion of G2 nucleus in path whereas the the 2B–D), in (Fig. bypassing cell the shape, of posterior helical a had nG,cmae ihtoei 1 Fg A.Temean The 2A). (Fig. G1. 6.8 in 8.9 was G1 those in 6 cells with of albeit parasites length compared small, in cytopharynx cytopharynx a G2, the noticed of We length in 2007). an the al., in in et were increase (Elias significant, (1N1K2F) G2 flagella of one two we stage and early and one thus, with kinetoplast kinetoplast epimastigotes one cycle; one whereas nucleus, G1, nucleus, cell in assuming were cycle, one (1N1K1F) of cell flagellum with of stages stage epimastigotes their different on that based in cells these cells classified of mixture h egho h yohrn n3 el.Tema cytopharynx mean The cells. 32 was in cytopharynx length the of length 1). the Movie along material cytopharynx supplementary the 1; of we (Fig. the length diameter However, a its only the and cells. cytopharynx in filled the all variation it along considerable in concentration as HRP prominent of suitable, sites it observed be made to and FIB-SEM proved the in cytopharynx tracer structure on this The and of images. recognition cytopharynx, 1991) the the of facilitating Souza, lumen thereby would (de de the it work endocytosis, through and fluid-phase homogeneously previous for distribute our Soares marker a on as 1978; based that, assumption was al., tracer et a Souza as HRP of choice irsoy(orsMdio ta. 02.W sdFBSMto FIB-SEM used electron We (which less scanning 2012). al., by specimen et surface with Medeiros a exposed (Soares newly milling) microscopy of serial albeit the ion-beam of focused surface the imaging cells, using with the microscope, combines the of of from inside It happens material reconstruction number tomography. of new 3D electron larger removal powerful rapid than a the resolution allows contrast, in FIB-SEM By organelles cells. of of technique number significant ARTICLE RESEARCH lcrndnesraeca unn rmisd h flagellar the this an inside 2228 In from by running opening. characterized the coat cytostome domain, see surface to membrane the electron-dense possible preoral-ridge was of it entire region epimastigote 1.5-nm flagellar positioned the the each transversely of slices, part and comprised virtual pocket volume 597 reconstructed adjacent in The seven thick. of resulting tomograms and sections, joining by fixation 200-nm produced of was 2) speed Movie the increase destabilization. medium membrane to added incubation and 2009), we cytoskeleton the minimize al., minutes, 10 to et for directly (Gadelha (Tf–Au)] the glutaraldehyde gold of [transferrin isothermal tracer colloidal architecture endocytic we the to the up complex, electron preserve complex, coupled taken had the To the cells resolution of after cells. of complex, parts different length different high from of long taken tomograms used the several to analyzed we Owing cytostome- tomography. the complex of organization cytopharynx ultrastructural the understand To cytostome- complex the cytopharynx of organization ultrastructural The 28 at minutes 5 for (HRP) peroxidase taken had horseradish that epimastigotes up of cytopharynx entire the reconstruct ..,weesclsi 2hdama yohrn eghof length cytopharynx mean a had G2 in cells whereas s.d.), he lc-n-iwsre fFBSMwr sdt measure to used were FIB-SEM of series slice-and-view Three h is oorm(oo1 i.3 upeetr material supplementary 3; Fig. 1, (Tomo tomogram first The m m 6 1.4 , m m( 8 m Fg A.I hs ape,teewsa was there samples, these In 2A). (Fig. m n 5 6.Mroe,drn 1tecytopharynx the G1 during Moreover, 16). m m 6 1.9 m m( ˚ .The C. n 5 16; mgn,b I-E,o h elsonhr a efudi supplementary in found be can here 1. shown Movie cell material the 1 of bars: FIB-SEM, Scale by flagellum imaging, (white). F, pocket mitochondrion; flagellar M, FP, complex; (orange); Golgi the G, (red, (green); reservosomes kinetoplast R, A–F cell 1–6). in of the (arrows indicated organelles of model cytopharynx lysosome-related model the the 3D in of A represented regions (G) the are cell. respective of the The end A–F. of Distal in posterior (F) shown the respectively). at 5, 6) structure and (arrow this 4 cytopharynx of (arrows diameter length the uneven its of the along portion 3). more distal and once 2 HRP-filled highlighting (arrows the cytopharynx, diameters showing an different sections showing with Transversal cytopharynx regions (D,E) the two of and section a lumen longitudinal small cell, HRP-filled a A and the (C) lumen of 1). empty (arrow posterior an diameter displayed the of cytopharynx Towards anterior the (B) of the HRP. section at of transversal (arrowhead) devoid cytostome was The cell (A) the 1–6), posterior. arrows showing to (white cell anterior complex a from cytostome-cytopharynx of the images of of portions Sequence different (A–F) of FIB-SEM. length using the along imaged accumulation HRP and cytopharynx diameter the in Variation 1. Fig. ora fCl cec 21)17 2723 doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal aaie hthdtknu R o iue were minutes 5 for HRP up taken had that Parasites . T.cruzi pmsioe) ,nces(le;K, (blue); nucleus N, epimastigotes); m .Tecomplete The m.

Journal of Cell Science EERHARTICLE RESEARCH irtblswsfuda oeatro oiin lsrt the to closer position, anterior more a 2 at 237 these found of slices Tomo end was virtual flagellar-pocket microtubules the at from Therefore, appeared respectively. generated Q4 205, and and ( model Q3 tomogram the already microtubules the of were contrast, slice quartet last in the the from in Q2 seen present and Q1 from As microtubules quartet 4A), 1 Q4. the (Fig. from Tomo to microtubules in the Q1 flagellar-pocket as numbered the to We not relative that, was plane opening. microtubules transversal noticed same these the of We in end found flagellar-pocket 4A). the (Fig. 3G), (Fig. of ridge path end the preoral one follow have the and that membrane clearly microtubules flagellar-pocket could four the we the slices, underneath 4), of virtual (Fig. existence 321 tomogram the 2; this (Tomo observe underlying In cell thick). different 3.14-nm a of in each region same devoid the of membrane a cytopharynx leaving 3I). cytopharynx, the (Fig. the the microtubules around of accompanied shape were microtubules ‘gutter’ side also of a sets body and in just two arranged cell These initiated membrane 3B,G,I). microtubules the (Fig. cytostome three cytopharynx inside was of the deep set microtubules different below going four purple then A and 3G–I). 3A–D), of (Fig. (Fig. cytopharynx 3E,F, set Fig. domain the membrane a lining this brackets; the cytoplasm, to underlying the close observed purple position In a 3C,D, surface). to (Fig. brackets) purple cytostome 3A–D, (Fig. pocket ecnimdteefnig yaayigascn tomogram second a analyzing by findings these confirmed We z sienme 2) By 321). number -slice noealve ftemdlgnrtdfo h reconstructed the from generated model gives 5A the Fig. of nm. view 3.14 of overall thickness an slice a with cytopharynx, the te ebaoseeet ncoepoiiyt the to tomogram. the of proximity edge the on close unambiguously were not they could because in them, we find identify although could elements 4E,F), we (Fig. associated cytopharynx cytoplasm, membranous of the devoid In other is 4D,G,H). membrane close, (Fig. the cytopharynx (i.e. very microtubules) cytopharynx the the endocytic observed of where an side to ‘naked’ was side tomogram, T1 the to this first particles fused, In by not Tf–Au although 4F,I). The on (Fig. followed containing cytopharynx 2 4E,F). vesicle the Tomo (Fig. 4E), of around (Fig. end microtubules cell microtubules one Q3 five projecting leaving the microtubule 4F), these thinner, (Fig. of was became interior of disappear quartet cytopharynx the the some the from towards Q4 as cytostome microtubule Interestingly, disappeared to the closest to 4D–F). T3, the to (Fig. close T1 being numbered tomogram were T3 microtubules with this its and 4A), in triplet (Fig. opening observed microtubule The 4A). first (Fig. was pocket flagellar the of opening rcs httkspaei h yohrn.Ti ol ebest be 3.62 covers could which This 5), cytopharynx. (Fig. endocytic 3 the the Tomo in in of place illustrated architecture takes the has that for cytopharynx process the consequence along important microtubules cytopharynx an the the of of arrangement side ‘naked’ The by the occurred along Tf–Au budding tracer vesicle endocytic the of Internalization ora fCl cec 21)17 2723 doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal oad h neiro h el K, cell. the of (*) anterior structure the this in towards bend a G2-phase showing in cells cytopharynx the of 3D models (E–G) White cytostome. cell. the the arrowhead, of posterior blue), the (N, reaching nucleus the around helical path a followed cytopharynx The G1-phase cells. representative three in (Cy, pink) cytopharynx the of models 3D * (B–D) test. two-tailed by analyzed unpaired an using of length 8.9 cytopharynx mean a had was G1 in 6.8 cells of length cytopharynx was analyzed 8.0 were cells cytopharynx all mean when The length cycle. phase G2 cell early the and of G1 from cells in and (Total) analyzed cells all of in cytopharynx epimastigotes the of of length analysis the Morphometric (A) for minutes. HRP 5 endocytosed had that cells 32 in the complex of cytostome-cytopharynx length the reconstruct measure to and able were we microscopy shape. and cytopharynx length of Analysis 2. Fig. ieols gen.Saebr:0.5 bars: Scale (green). kinetoplast m m m m m m 6 6 6 1.4 1.9 2.0 m m m .Tedt a analyzed was data The m. G2 in cells whereas m, m( 6 t ...Temean The s.d.). ts n post- and -test yuigFIB-SEM using By m nlnt of length in m P , 0.01. .cruzi T. 2229 m m.

Journal of Cell Science EERHARTICLE RESEARCH opooyadcnann ag ubro odparticles tubule-vesicular gold of a 2230 number large with a dilated containing tracer this compartments to and Close the morphology tomogram. endocytic the cytopharynx of the where edge of portion, the portion at expansions dilated sectioned a was 5A,G, was forming Fig. cytopharynx In the concentrated. homogeneous, inside particles Tf–Au not of distribution The inside distributed cytopharynx evenly the not were particles Tf–Au the the at cytopharynx. found and the with frequently of nm, portion more 90 distal be aligned of the to diameter seemed vesicles vesicles approximate gold-containing All with tracer the fusing had this membrane. or cytopharynx from without cytopharynx budding or clearly the observed 5E,F) were (Fig. 5C,D) Importantly, Tf–Au (Fig. 5B). complex (Fig. containing Golgi cytopharynx the the vesicles that to length proximity noticed the close We in along 5A,B). was be cytopharynx, (Fig. to the structure seen of this be side of could naked particles, the Tf–Au with vesicles) with ar aligned blue seen vesicles) be (light (silver can (bl not loaded quartet membrane or vesicles, and cytostome-cytopharynx triplet the uncoated microtubule of Many the side I, tomogram. ‘naked’ In (pu microtubule-free o ridge cytostome. a surface preoral of leaving the the base cytopharynx, of to the the edge adhered nm. at (white) of 200 (dots) initiates pocket path bars: Th Tf–Au flagellar that the Scale of (F) the (green) following presence arrow). arrowhead). and triplet and (blue) the white microtubule opening quartet and (rectangle, the microtubule cytostome arrowhead) pocket with The Model the white flagellar model. along (E) (pink, the the seen, mitochondrion. cytostome from of appea be M, views the (purple) which can flagellum; Cytoplasmic showing ridge arrows), F, (G,H) position, preoral (blue B). ridge. different the quartet preoral in a of the microtubule arrows from emergence underlying (green observed the the triplet model and and microtubule same region (Cy), a flagellar-pocket cytostome with the the cytoskeleton. together showing to the cytostome-cytopharynx Tomo1 (FP) with the pocket association to four flagellar its support of the and mechanical Set from complex (A–D) extending minutes. cytostome-cytopharynx brackets) 10 the for (purple of Tf–Au organization endocytosed had 3D that the epimastigote 1, Tomo 3. Fig. z sie on rmpseirt neirrgoso h el hwn h roa ridge preoral the showing cell, the of regions anterior to posterior from going -slices el ehv xmnds a,atr21 iue fincubation all of minutes In 2–10 6B,C). after (Fig. far, so ended examined to cytopharynx have seemed we the each they cells from and after (two cytopharynx, even microtubules the continue around four observed only were point, set) this lysosome-related At (a 6C). reservosomes (Fig. the of to organelle close very some fixation. positioned out chemical rule to techniques, cannot inherent we variation the different cells, osmotic of of different using contribution in length its observed and and along tracers been diameter different diameter in have in variations cytopharynx varies the cytopharynx Although engaged is had regions length. the it that when inside cells endocytosis, that, by in shown clusters have observed in separated we we Therefore, as in HRP. 6A,B), cytopharynx, up (Fig. taken gathered tracer the the particles of of devoid Tf–Au thick). portions nm find 1.9 enlarged each slices, could virtual 239 We 4; (Tomo cytopharynx the endosomes 5G). early (Fig. the organism to this correspond of should and observed be could h yohrn ne nalrebgsae structure, bag-shaped large a in ended cytopharynx The of portion final the covers that tomogram serial a shows 6 Fig. ora fCl cec 21)17 2723 doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal .cruzi T. pmsioe)a h otro ftecell the of posterior the at epimastigotes) eiltmgah fan of tomography Serial pearrow) rple of st give to rs e ound ack f

Journal of Cell Science EERHARTICLE RESEARCH ofe,16;Lcml ta. 09 iadDa tal., et and Girard-Dias 2009; Taylor al., 1969; et (Preston, a In Lacomble 2012). flagellar trypanosomes to 1969; the correspond other surrounds Godfrey, microtubules that in microtubules these pocket of whether quartet both question to led conserved complex common to is cytostome-cytopharynx the that us and quartet pocket microtubule flagellar a the of existence the The in contained of pocket flagellar were The dilation. tracers bag-shaped a the in ended tracers, which cytopharynx, endocytic flagellum; F, new po and orange, flagellar micro with ; E five the flagellum in only at F, cytostome) arrow complex; microtubules the green Golgi of to dark G, positioning (relative mitochondrion; the cytopharynx the M, and the which Other kinetoplast; nm of arrow in H. K, portion 200 blue A, and shape. distal bars: light in most G half-moon Scale The the a model in flagellum. disappear. At in the model (I) old (F) arranged of the visualized. triplet structure, view in better runn this the Back arrowh be yellow thinner, around from can becomes (black (G) in seen T1 cytopharynx cytostome vesicle microtubules. seen the the and a these be As and (E) and F). of also ridge quartet and cytopharynx end preoral can E the lini the very vesicle in from seen around the (asterisks This Q3 be microtubules cytopharynx indicate cytopharynx. t can microtubules the of respectively, the accompanies of arrows) cell, set of side also full (blue the naked side and the into quartet the naked cytostome cytostome see to deeper microtubule the the close the can The observed to accompanying the we of (B) were close cytoplasm, of D opening compartments A. dots) the In beginning the membrane-bound in (black into contour). the to indicated particles bends (purple showing adjacent regions Tf–Au ridge then 2, position the Tomo preoral containing and a representing of the (purple) from tomogram model and ridge originates the (Cy) 3D preoral green) of cytopharynx the the T3, slices of of to Virtual View path (T1 (B–F) (A) the triplet cytopharynx. cytopharynx. follows microtubule the that A along blue) (pink). microtubules Q4, cytopharynx of to positioning (Q1 cytopharynx. the quartet the showing microtubule along minutes, microtubules 15 of for distribution the Tf–Au 2, Tomo 4. Fig. irtbl quartets microtubule h t mcouueqatt,adi opie h only the membrane. comprises flagellar-pocket it the and with quartet), associated (microtubule microtubules MtQ the T . brucei hscnevdqatti fe eerdt as to referred often is quartet conserved this , .cruzi T. pmsioe a two has epimastigotes eiltmgaso neryG-hs 112)eiatgt hthdtknup taken had that epimastigote (1N1K2F) early-G2-phase an of tomograms Serial irtbl otx erteoeigo h lgla pocket flagellar MtQ. the subpellicular the of to the flagellar corresponded into opening clearly the This inserted the 7A–E). surrounded and (Fig. near pattern body, flagellar-pocket cortex, helical basal microtubule a the the in to pocket underlying close different originated microtubules two of The of 7). presence membrane of Fig. the 5, demonstrated 1.58- quartets 7E), (Tomo each (Fig. slices, pocket thick virtual 771 nm flagellar of consisting the volume, reconstructed of (Gadelha volume pocket flagellar entire the of components lumen 2009). extracellular the al., which to pocket et access through flagellar the gain channel in can a domain forms membrane a that underlies MtQ The epromdasra oormo elta oee the covered that cell a of tomogram serial a performed We ora fCl cec 21)17 2723 doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal .cruzi T. pmsioe.Oeo h quartets the of One epimastigotes. uue were tubules ih blue, light gthe ng cket, he 2231 - ead) ing

Journal of Cell Science EERHARTICLE RESEARCH 2232 oktt h otro eino h el(i.8A–C). (Fig. cell the microtubules of cytostome the region flagellar of the shape posterior from arched the quartet the microtubule Interestingly, cytostome to different the two pocket follow the and identify and quartets could we these epimastigotes we In for these (TEM). cytoskeleton, preparations, cytoskeletons microscopy electron of whole-mount investigate entire transmission by resulting observation the To the membranes stained of cytopharynx. negatively the context parasite the the the detergent-extracted into in with extended preoral microtubules microtubules the along of these the that body, of then accompanied axis and path anteroposterior ridge, Their the 7A–C,E). to found (Fig. was relative was microtubule cell position each microtubules different of end these a to the at of Also, anterior area. end but body membrane, One basal the flagellar-pocket 2. the and below 1 positioned Tomos in shown h irtblso h te ure ersne h ones the represented quartet other the of microtubules The ihu h yohrn ebae ecudcutfive even count nucleus, cell, could the the posterior We of posterior immediately membrane. the microtubules cytopharynx to cytopharynx path the cytopharynx entire without the the each is along with cytostome associated the the other of closely where from images one remained the microtubules nucleus to These portion relative the found. negative-staining cell of the of anterior plane The side our the opposite reached the C). from Also, always nucleus microtubules that posterior. the and around confirmed to path B the helical of appeared anterior a structures in followed the microtubules cytopharynx compare cytoskeleton 8, cytostome- Fig. where cytostome-cytopharynx the (in of ‘bare’ be total areas cytoskeleton, comparatively to after other appeared to even region cytopharynx relative cytoskeletons, This complex 8A,B). the structurally (Fig. ridge in preoral extraction the preserved membrane of region always predicted the was underneath pass they as ora fCl cec 21)17 2723 doi:10.1242/jcs.135491 2227–2237 127, (2014) Science Cell of Journal nooe,lae ihT–u(yellow) Tf–Au with loaded endosomes, early the tubule-vesicular probably of compartments, series A Tf–Au. filled portion with distal dilated a presents cytopharynx (Cy) The (G) cytopharynx (arrow). the membrane with fission) undergoing or (i.e. fusion contact be direct also in can seen Tf–Au A with (E,F) loaded D. in vesicle model 3D the is in This represented membrane. cytopharynx contact the direct with in an (arrow) shows vesicle tomogram empty the of a image C, virtual In (C,D) vesicles). (silver others empty whereas were vesicles), blue (light Tf– Au contained cytopharynx the vesicles to the aligned of the Some to (B) proximity cytopharynx. its show also to was modeled (red) of complex portion Golgi A the cytopharynx. silver) the and to blue adjacent (light vesicles a of and series blue) and (green associated microtubules its with (pink) represents cytopharynx model the The (A) for minutes. Tf–Au 15 up taken had an that of epimastigote cytopharynx the of portion distal the of reconstruction Tomographic cytopharynx. membrane the active of the domain 3, Tomo 5. Fig. ADG;10n (E). nm nm 100 200 (A–D,G); bars: Scale cytopharynx. flagellum. the Orange, to close seen are

Journal of Cell Science EERHARTICLE RESEARCH hssrcuei t nieywe sn nyaaye of analyses only using when entirety following its in difficulty in the to due structure this was gained, at This endocytosis was this cycle. of life site structure the main of this the cytostome-cytopharynx stage as about importance great the knowledge its despite further of little description very of initial complex the Since DISCUSSION the of end the CaCl towards with treatment after depolymerize four to to bars: reduced Scale contour). 8A–C). the blue were (Fig. around (R, cytopharynx observed reservosomes be the these can and set) cytopharynx each and the of of (two end (C blue) microtubules the (arrowhead). (B (light four between cytopharynx Tf–Au. Vesicles Only of proximity of arrows). cytopharynx. end the concentration (white the the Note of beyond concentrated around (inset). nm. regions extend is microtubules cytopharynx arrows, 200 microtubules Tf-Au of the cytopharynx White where distribution of remaining entirety. (pink), the the portion its cytopharynx that showing distal in the Note model almost cytopharynx. in the seen the diameter of be to cytopharynx. enlarged view can close the of observed (Cy) of regions be cytopharynx lumen the also the the can showing of in 5 portion Tomo clusters final of in the model found that are so particles rotated Tf–Au plane, 4, Tomo 6. Fig. lhuhteedctctae a rsn nteextracellular vesicles. the flagellar-pocket the in inside that, present found was was Note tracer in tracer no membrane. seen milieu, endocytic flagellar-pocket also the the was diameter although with 3), in nm Box Fig. contact 274 S2D,E; direct measuring material Fig. vesicle, material (supplementary large (supplementary A (supplementary structure 2). diameter precursor Box tubule-vesicular a S2A,C; resembled in other a the nm and 32 of 1), Box of S2A,B; a Fig. in represent vesicle material to shown budding appeared them are small of 5 one membrane; Tomo flagellar-pocket in could pocket we S1. Fig. flagellar cytostome- microtubules material the the supplementary the to related near of not observe All are model they microtubules. the because in cytopharynx 7, included microtubules Fig. not were cytoplasmic in These shown described 2012). al., recently et (Girard-Dias other found also salt upon disappeared treatment. microtubules shown). subpellicular the (not expected, cytoskeletons cytopharynx As salt-treated the of in lengths remained varying microtubules of the sections to region, Distal treatments. cytostome salt these to resistant also was cytoskeleton Gl,19) ecnimdta hswsas h aein case the also CaCl was mM 60 this or 8D) that remain (Fig. MtQ confirmed the cruzi We and T. microtubules 1999). flagellar the (Gull, whereas salt, of In nTm ,torr naiain eeosre tthe at observed were invaginations rare two 5, Tomo In have we 5), (Tomo pocket flagellar the of reconstruction our In .brucei T. pmsioe ytetn yokltn ih1MNaCl M 1 with cytoskeletons treating by epimastigotes .cruzi T. h upliua irtbl osti known is corset microtubule subpellicular the , pmsioe Mle n en,1969), Deane, and (Milder epimastigotes 2 Fg E.Ipraty h cytostome the Importantly, 8E). (Fig. 2 rhg concentrations high or eeuigsra lcrntmgah feiatgts where epimastigotes, of presented tomography analysis electron the requiring serial of structure lacking, using the was here of link description clear physical a (Okuda this Nevertheless, co-workers and 1999). Okuda al., the by et physical and suggested pocket been a had flagellar of cytostome the of existence between The link these unclear. cytoskeleton-mediated hallmark of around remained origin arrangement cytopharynx and the the positioning a However, exact 1969) 1996). their al., and Preston, microtubules et is 1960; (Attias Novikoff, bodonids and complex and (Steinert trypanosomatids cytostome-cytopharynx the investigation. detailed under during now complex are cytostome-cytopharynx division the involved cell of events duplication The the the in division. of cell in changes remodeling for the the preparation reflect that in G2 suggest cytopharynx in strongly length we and of shape capacity this, cytopharynx endocytic on not the Based was in endocytic cells. 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Parasites METHODS AND MATERIALS noyi rcs nti neetn n eeateukaryotic relevant efficient and highly interesting the this understanding in model. and of process cytostome-cytopharynx possibility the endocytic so of the used structure spatial the with opened of complex been features the new compared and have revealed of long analyses that epimastigotes, 3D analysis high-resolution observation The the in far. of for methods endocytosis adequate conventional of more be dynamics to proven the towards cell. vesicles or compartments the organelles of directing speculate cell in posterior could role cytopharynx. internal a one play the the microtubules, any they of that these observe towards end with not connected the continued directly did past running and we cell, Although present four the cytopharynx, of the still of posterior cytopharynx portion were bud final the the vesicles microtubules At along where cytoplasm. points’ form the ‘exit into clusters preferential these characterize cargo. the might where of insoluble nature region the an of tracer, independent The fluid-phase is Tf–Au, feature a this peroxidase, that using with showing as well when as tracer, observed particulate was cytopharynx. cytopharynx the of that end assumption the the to at leading exclusively for thus occurs mistaken structure, endocytosis been this of have end might distal length the its enlargement along an cytopharynx observation, the of of plane were the sections, on TEM ultrathin Depending only from analyzed. cytopharynx, where the publications of previous images in endocytosis. 2D missed in been engaged have endocytic are might of they This enlarged that clusters suggesting of contained strongly regions cytopharynx tracers, cytopharynx the the Also, the length. along that its diameter serial along showed different diameter series in of varies analyses was FIB-SEM our it and contrast, description, tomograms By this cytopharynx. end on enlarged the the Based of at exclusively 1978). Deane, occurred al., and endocytosis that et (Milder assumed structure Souza bag-shaped de a roughly 1969; a in – with proper terminates cytopharynx element the which – tube-like length its a along – by diameter homogeneous opening followed the funnel-shaped – a Thus, cytostome having the the tracer. as now, described Until endocytic length. was its cytopharynx along the varies cytopharynx contained the was four of cytopharynx diameter and only the where enlarged locations, these cytopharynx often At the found. in of are often regions microtubules are the to compartments endocytic proximity of is other close this that end compartments. that noticed other fact with have the content the We of to exchange at of contribute site cell. preferred to found the the likely are is inside which deeper microtubules cytopharynx, goes fewer and thinner Therefore, becomes structure 2005). this al., et Vieira 2000; al., et (Porto-Carreiro rm35dyodclue eeue o h experiments. the for used were cultures 3–5-day-old from ncnlso,te3 eosrcinwr rsne eehas here presented work reconstruction 3D the conclusion, In the along clusters in cargo of aggregation the Interestingly, as terminate cytopharynx the around microtubules the of Some pmsioe rmcoeD2cwr utvtdi liver in cultivated were Dm28c clone from epimastigotes ˚ .Epnnilpaeparasites Exponential-phase C. + -ATPase odprils(fA)fr1 r1 iue;o R o iue,both minutes, 5 for to HRP or coupled minutes; sodium transferrin 15 28 or contained incubated mM 10 at that [10 were for FCS) (Tf–Au) Cells particles 7.2]. saline (without gold pH medium NaCl, phosphate-buffered RPMI (w/v) in 0.9% in with buffer twice phosphate washed were eoeteedctctae n rcse o rnmsinelectron transmission for processed and tracer to (TEM). PBS microscopy in endocytic twice washed the the were to cells remove directly the 2.5% fixation, of After medium. concentration incubation final a to glutaraldehyde isothermal bevduigaTecnai-G a and water using in aurothioglucose observed (v/v) 0.7% with stained negatively were Grids H76 o 5mntsi h akada omtmeaue fe this After temperature. room buffer, at and Tris-HCl dark in the (Sigma) in DAB H minutes incubation, 15 mg/ml incubated for 0.5 were 7.6, HRP containing up pH solution taken had a that in cells TEM, for processing Before 3-3 1500 at centrifugation by collected were Parasites medium. culture in experiments fluid-phase Endocytosis mg/ml 1 as of used concentration was final a Sigma) (Roth, described at previously (HRP, described tracer as peroxidase as particles Horseradish prepared gold St to 1983). Sigma, were coupled (Tf, was holotransferrin nm) MO) Bovine 8–10 Louis, 1985). Geuze, (Au, and (Slot previously particles gold Colloidal tracers Endocytic o lgmn ftetle iw.Snl-xstl eis( a series and software tilt Xplore3D Single-axis using Tecnai-G samples markers views. from fiducial produced tilted as were were used the increments) nm) be of to (10 sections, alignment particles the for post- of gold surfaces stained Colloidal both and on above. deposited grids described slot ribbons as copper These embedding formvar-coated were above. onto sections described as collected serial prepared were thick blocks 200-nm TEM of from produced ribbons tomography, electron For tomography Electron mM 1 PIPES, mM (100 buffer PEME in NP-40 MgSO (v/v) 1% grids, with formvar-coated glow-discharged extracted onto mounted were Epimastigotes cytoskeletons whole-mount of Preparation Tecnai-G uranyl a resin. using (w/v) by epoxy observed 5% were in and with embedded citrate, post-embedding lead and and stained acetate series were acetone sections an Ultrathin in (w/v) three dehydrated in 1% washed After were water, samples of the post-fixation, osmium minutes. post-fixative Following minutes. solution the 5 in 3 again for a minutes, solution for incubated 40 were in cells water the for incubated water, in in washes 7.2] then Sigma) pH and buffer, (TCH, water thiocarbohydrazide cacodylate in M twice 0.1 washed in mM 5 chloride and ferrocyanide potassium calcium solution (v/v) osmium 0.8% post-fixative tetroxide, osmium- osmium a (v/v) in an [1% incubated were using a cells post-fixed the Briefly, isothermal Following were 1984). Rutherford, in cells and above). (Willingham fixed the protocol (OTO) or described buffer, thiocarbohydrazide-osmium temperature, cacodylate as room in wash endocytosis, at cacodylate hour (after M 1 0.1 glutaraldehyde for in glutaraldehyde 7.2, (v/v) pH 2.5% using buffer, by fixed were Cells microscopy electron Transmission rprto fsl-rae yokltn,giswr nuae n1MNaCl M 1 the in CaCl mM For incubated 2003). were 60 or grids al., cytoskeletons, et salt-treated (Parussini of PEME preparation in glutaraldehyde (v/v) 2.5% with irsoeoeaiga 0 kV. 200 at operating microscope prtn t20k n ope oa4k a to coupled and kV 200 at operating h el eekp ntedr o ute 5mntsa room at minutes 15 further a for dark and washed the TEM. then were for Cells in processed 1966). 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TEM conventional so like inverted looked contrast they that their had of image images removal After electron beam. the slice- back-scattered ion for the a focused chosen capturing, the create was with surface nm To specimen 25 mode. the from of a lens material size immersion and step the a kV series, in 1–5 image Back-scatter pA and-view of surface. 4000 voltages the of accelerating smooth current at and beam scanned polish recorded was to that were beam nA images trench ion 3–7 electron the a step, of create second current the a to In using specimen. kV the 30 50–150- into 7– Usually, of cut (typically cross-section. were currents voltage the accelerating beam of high an viewing at enabled at made and was nA) cut two 20 in coarse introduced was a cut cross-sectional First, A stages. beam. the to parallel was stage otiigtesml a rme oaprmdlsae n t surface its block and resin shape, the pyramidal of a top to minutes trimmed The 5 was above. for sample described HRP the as containing up TEM taken for had processed that were cells FIB-SEM, by using observation For segmented microscopy electron manually scanning models. Focused-ion-beam 3D were produce to slices used also JOIN Virtual was the which using IMOD. series) 3DMOD, a in in sections (of window followed tomograms adjacent ETOMO, of using joining performed by was back-projection R-weighted by ARTICLE RESEARCH tis . omr,R .add oz,W. Souza, de and C. R. Vommaro, M., Attias, E. Angelopoulos, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135491/-/DC1 online available material Supplementary material Supplementary Cientı Desenvolvimento de Nacional Conselho Tecnolo by supported was work This paper. the Funding of drafts on N.L.C.S. commented and authors C.L.A. All data. paper. interpreted N.L.C.S. the and and wrote experiments W.S. designed experiments. and performed conceived and designed J.C.V. and C.L.A. contributions Author interests. competing no declare authors The interests Competing Leite Moreira F. manuscript. Flavia the to of grateful reading Ju most Sergio are Biofı Luis We de Moreira and Brazil). (Instituto Barros Brazil) Janeiro, de Janeiro, de Luis de Rio Thiago Rio (Inmetro, of Bioimagem, assistance de technical Nacional the (Centro for grateful are We Acknowledgements injector was gas 52 stage to specimen a sample The tilted using chamber. entire specimen layer main the platinum the imaging, inside 3-nm-thick located SEM system a with and coated milling was to surface Prior imaging. detector SEM electron secondary in-lens for an focused-ion-beam and gun for field-emission a source and gallium-ion milling, a with column. microscope equipped dual-beam the microscope 600 Company) of Nanolab (FEI Nova the surface a the using to by with imaged perpendicular paint, were Samples upwards, silver using pointing stub block SEM resin an to glued block then The knife. was diamond conventional a using sectioning by smoothed was ih eApr a Fundac Amparo the de from Filho E-26/102.850/2012] number Nu [grant Programas Estado (FINEP), Projetos e Excele Estudos de Financiadora J.C.V., and Aperfeic iesoa eosrcino th of (Kinetoplastida:Bodonidae). reconstruction dimensional .Protozool. J. ni POE)[rn ubrE2/1.7/00 n init oNosso do Cientista and E-26/110.576/2010] number [grant (PRONEX) ˆncia gc CP)[rn ubr426/022,Coordenac 472262/2012-2], number [grant (CNPq) ´gico ¸ aet ePsold Nı de Pessoal de oamento ˚ n xoe otefcsdinba ota h ln fthe of plane the that so beam ion focused the to exposed and 17 sc alsCaa ih,Rod aer,Bai)frcritical for Brazil) Janeiro, de Rio Filho, Chagas Carlos ´sica eqian saod i eJnio(FAPERJ). Janeiro de Rio do Estado no Pesquisa ` 39-51. , 17) elclrmcouue ntefml Trypanosomatidae. family the in microtubules Pellicular (1970). elSrc.Funct. Struct. Cell vlSpro CPS coasist C.L.A to scholarships (CAPES) Superior ´vel relvn rtza oosp. Bodo protozoan free-living e 21 19) optraddthree- aided Computer (1996). 297-306. , ¸ a oCro Chagas Carlos ˜o m -ietrenches m-wide ¸ a ode ˜o ce de ´cleo fc e ´fico ´nior kd,K,Etv,M,Sgr,E .adBjvy .T. A. Bijovsy, and L. E. Segura, M., Esteva, K., Okuda, idr .adDae .P. M. Deane, and R. Milder, ee,H n eSua W. Souza, de and H. Meyer, K., M. Shaw, K., Martı M. Morphew, C., Gadelha, S., Vaughan, S., Lacomble, R. J. McIntosh, and N. D. Mastronarde, R., J. Kremer, K. Gull, J. M. Karnovsky, and Jr. C., R. Graham, Alca W., Girard-Dias, Gull, and J. N. Severs, R., J. McIntosh, M., Morphew, S., Rothery, Freymu C., A., Gadelha, R. Mortara, P., F. Faria, de P., J. Cunha, da C., M. Elias, eSua . eCrah,T . ecio,M n hai E. Chiari, and M. Benchimol, U., T. Carvalho, de W., Souza, de P. E. Camargo, ilnhm .C n uhrod .V. A. Rutherford, and C. M. Willingham, iia . olf,P,Lo . uh--iv,N . eSua .and W. Souza, de G. L., D. Russell, N. and P. Cunha-e-Silva, Webster, S., Luo, P., Rohloff, M., Vieira, Garcı F., Parussini, aauaaua . Ueda-N C., VataruNakamura, B. A. Novikoff, and M. Steinert, K. Miranda, and H. Barrabin, C., Jiao, W., Souza, De C., L. Medeiros, Soares W. Souza, de and J. M. Soares, J. H. Geuze, and W. J. Slot, S. Schenkman, and E. Freymuller-Haapalainen, C., T. Ramos, M. T. Preston, alr .E n ofe,D G. D. Godfrey, and E. A. Taylor, J. Roth, N. Cunha-e-Silva, and W. Souza, De K., Miranda, M., Attias, I., Porto-Carreiro, Souto-Padro W., Souza, de F., P. Pimenta, conorhini. nrmmrn atce.Actceia n reefatr td nculture in study and freeze-fracture components cruzi. and Trypanosoma coat of cytochemical surface forms of A distribution particles. the intramembrane in differences Topographical by revealed trypanosomes in cytoskeleton tomography. microscope associated electron and pocket K. flagellar Gull, the and R. J. McIntosh, IMOD. using data image three-dimensional of Microbiol. kidney: mouse of tubules technique. proximal new 291-302. the a in by cytochemistry peroxidase ultrastructural horseradish injected of tomography. electron 138 and methods cryopreparation using K. Miranda, trypanosomes. African 17425-17430. in cytoskeleton the by K. cycle. cell S. Schenkman, iseclue fpgeteihlu rmtecikeby.Utk fmelanin of Uptake embryo. parasite. chick the the from by epithelium granules pigment of cultures tissue rpnsm rz:utatutrl yohmcladfez-rcuesuisof studies freeze-fracture uptake. and protein cytochemical ultrastructural, cruzi: Trypanosoma Media. Liquid 6 in Trypanosomes Metacyclic of thiocarbohydrazide-o noyi aha fTyaooacruzi. Trypanosoma of pathway endocytic R. Docampo, complex. flagellar the with Parasitol. associated is epimastigotes cruzi Trypanosoma ehd oehnemmrn otatadpeevto nclue cells. cultured in preservation and contrast Cytochem. membrane Histochem. J. enhance to methods aaio.Today Parasitol. backscattered field and resolution secondary high by using cruzi microscopy imaging. Trypanosoma electron electron in scanning cytostome emission the of Visualization with infected microscopy. electron erythrocytes beam-scanning multiple ion ONE focused of PLoS by architecture nanoscale at 3D Plasmodium the Visualizing (2012). cruzi. Trypanosoma by LDL cytochemistry. multiple-labeling sections. thin on glycoconjugates of Laveran raiae Trypanosoma Mesnil. haemoflagellate & elasmobranch the of forms culture J. cruzi. Trypanosoma J. from Cazzulo, carboxypeptidase and L. Aslund, aiaintrypanosomes. salivarian Cytol. in Biochem. Biophys. Pinocytosis J. of Occurrence cycle. organelle division and cell the epimastigotes along cruzi distribution Trypanosoma of reconstruction dimensional in storage before network endosomal the early enters an cargo reservosomes. through pathway: passes endocytic and epimastigote cytostome label- cruzi replica-staining Trypanosoma (2000). fracture-flip, a cruzi: Trypanosoma survey. of fracture surface cell The 93-100. , nzPlm,A,DSua .adGnae-ols A. Gonzalez-Robles, and W. 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