1. Protozool., 2 5 ( 3 ) , 1978, pp. 287-292 0 1978 by the Society of Protozoologists

Mitosis in the Hemoflagellate Trypanosoma cyclops* PETER HEYWOOD and DAVID WEINMAN?

Division of Biology and Medicine, Brorvn University, Providence, Rhode I t land 02912 and School of E f d e m i o l o g y and Public Health, Yale University School of Medicine,

New Haven, Connecticut 0651 0

SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while a t the periphery of the nucleus condensed chro- matin is in contact with the nuclear envelope. The nucleolus fragments a t the onset of mitosis, but granular material of prcsnmp- tive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact lvith the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The hasal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed. Index Key Words: Trypanosoma cyctops; spindle microtubules; nuclear envelope; nucleolus; chromatin ; electron microscopy.

UCILEAR division has been described in Trypanosoma cruzi N ( 2 , 4), Trypanosoma equijierdum ( 14), Trypanosoma gam- biense ( l o ) , Trypanosoma rhodesiense ( 19), Tryfianosoma raiae ( 19) , and Trypanosoma uiuax (17). Although some specific differences do exist, it is now apparent that in its major features mitosis in the trypanosomes differs markedly from that reported in many other cell types including mammalian cells. As sug- gested Iiy Vickerman ( 18), these differences might be important in chemotherapy and hence study of trypanosome mitosis has a practical aspect. Investigation of mitosis in the trypanosomes is also of interest because it adds to our knowledge of mitotic mechanisms and might be useful in determining their evolutionary significance.

In this paper we describe the ultrastructure of mitosis in lab- oratory cultures of Trypanosoma cyclops Weinman and compare this with piil)lished accounts of mitosis in other trypanosorne specics. The usc of serial sections has made possible a more accurate description of nuclear structures and events.

MATERIALS AND M E T H O D S

Trypanmoma cyclops was grown on blood agar slants a t 25 C (20) . Cells Icere harvested 3-5 days postinoculation a t which time the culture was growing logarithmically and - 1-3% of the cells were in mitosis. Cells were fixed for 1 h a t 10 C in a 1: 2 mixture of Karnovsky fixative ( 1 I ) and of 0.1 M phosphate buffer, pH 7.0 (9) . After several rinses in buffer, cells were postfixed with a 1 % (w/v) solution of Os04 in phosphate buffer for 1-2 h at 0C. Cells were washed thoroughly in buffer, de- hydrated in an ethanol series, stained in hulk in a 1% (w/v) solution of uranyl acetate, and embedded in Epon. Ribbons of sections were cut with a diamond knife and collected on single- hole copper grids coated with Formvar and carbon. Sections were stained with aqueous uranyl acetate and lead citrate before being examined in a Philips 201 S electron microscope.

Figures 20-25 are cross sections of the mitotic spindle photo- graphed a t 100 kV from sections with an estimated thickness of '200 nm. All the remaining electron micrographs are longitudi- nal or obliquely longitudinal sections of nuclei photographed a t 80 kV from sections with an estimated thickness of 70-90 nm.

* This investigation was supported by Research Grant A1 09888 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, and a Biomedical Research Support Grant from Brown University. + Present address: Department of International Health, San Francisco Medical Center, San Francisco, California 94143.

T h e compositc line diagram (Fig. 9 ) was prepared by sepa- rately projecting the negatives of Figs. 3-8 onto a piecc of paper a t 81,000x magnification. T h e outline of the niiclens and the disposition of the spindle microtubules was succcssi\rely recordcd for each figure. For simplicity, the nuclear periphery in the resulting diagram was represented by a line of best fit. The diagram was photographically reduced to 35,000X magnification.

OBSERVATIONS

The ultrastructure of T. cyclops has been discussed at length elsewhere ( 9 ) . When grown on blood agar slants, this organism possesses electron-dense pigment I~odies (Figs. 2, 17-19).

T h e nucleus is situated immediately posterior to the kinetoplast and flagellar basal body (Fig. I ) . Although all 3 organrlles are located close to one another, there is no direct niorphologic participation of the basal body in mitosis. T h e interphasc~ nucleus is roughly cylindrical, ccith the diameter of this cylinder cquiilliiig 1.2-1.6 pin (see Figs. 2-5, 9 in Ref. 9 ) . T h e central region of the interphase nucleus is occupied by a single spheriral nucleolus, composed mainly of granular elements of diaineter 21-27 11111.

Electron-dense chromatin is seen around the periphery of the nucleus and is in contact with the nuclear envelope. Thc nicin- hranous structure of the nuclear envelope is not clcar in our preparations fixed in a mixture of Karnovsky fixativc and phos- phate buffer, but its location is defined by an electron-op;qiie zone that coinpletely encircles the nuclcus. This miic tn;iintains the same appearance throughout mitosis. In high niagnification electron micrographs dense spherical structures are distingnishable on the cytoplasmic side of this electron-dense zone; thc strncturc and location of these structures suggest that they rrprcsent i.ibo- somes attached to the outer nuclear nieinbrane.

T h e beginning of mitosis is marked by several changes in nuclear structure-spindle microtubules appear, the nucleolus fragments, and chromatin becomes less clearly defined. Figurt-s 3-8 are serial sections through a nucleus at an early stiige of mitosis. Figure 3 passes through one edge of the spindle, u.liile the convergence of spindle microtulmles a t one of the poles is evident in Figs. 5-7. Although the other pole is not reprcsmted in this series, it can be seen in the composite diagraiii of thc available electron micrographs (Fig. 9) that spindle microiuhules are approximately parallel to each other and ??;tend ;IS a single bundle from the nuclear periphery at one pole across the nucleus toward the other pole. Sections of nuclei a t earlicr stagcs in mitosis reveal a somewhat siinilar arrangement, except that the bundle of spindle microtubules is less compact, i.e. individual

287

288 MITOSIS IS T t y p n o s o m n cyclops

All figures are of 7'. cyclop.~ epin~astiqotes grcmn on Iilood agar slants. Except for Fig. 9, all figures are electron micrographs.

Fig. 1. Longitudinal section thrnugh the nuclear region o f an interphase cell. Note the close relationship between the flagellar basal hody (I)), kinetoplast ( k ) and nuclrus. .A nucleolus i n ) i n the center of the niicletrs, and condensed chromatin ( c ) are seen around the periphery of the nucleus. x 20.000.

Ohlique longitudinal section through daughter cells \\hich are heing separated by a division furrow. A flagellum ( f ) , hasal body ( b ) , kinetoplast i k ) and pigment l)odies (11) are evident. The spindle ( s ) and re-forming nucleolus ( n ) of the late division nucleus are seen in greater drtail i n Figs. 17-19. x W.000.

Fig. 2.

niicrotiil)nlcs a rc oftrn some distance froin thcir nearcst neigh- bors. In contrast, later stagcs of mitosis ha\.(, :I vcry compact arrangement of spindlP niicrotril)iilcP ( Figs. I 7-25 ) . Frequently these niicrotubiiles a re regnlarly arrangid ( Iigs. 21-23 ) and iiiteriiiicrotii1)iiIe 1)ridXrs ( 6 ; can .;oiiic*tiinrs l)r distin~iii.;hecl [Fig. 16).

During the tours(' of mitosis thc, niic~lcn.c elongatrs in a plane appioxiinatrly perpendicwlar t o thr lon~j tudinal axis of the cell. Thv cnd rrsult of this procrss is seen in Fig. 2, where the 2 daughter cclls are p s e n t i n ohliqiic longitiidinal section and the division fiirroiv separating thrni i.; approaching the isthmus I-rgion ( I!)) of the elongatrd I n t r division niiclrus. L41thoi~gh part of thr nucltws lies ont nf thc plane of section, it is apparent that the spindle c,xtcnds acinss thr 2 cells a~ right anglrs t o their longitudinal ;IXCS. T h c iiiitolic nlic~lrus o f T . r r i r i i has a 5iniilar orientation ( 4 ) .

In rarly and iiiiddlr phases of niitosis thc nucleu.; I)cconics an increasingly elongat(. c$indrical \triictiirf*. Evrntually the niid- rrgion of the nnclrns I)rc-omrs c.nnslrict(d rrsrilting in a dnnih-liell iipprariince in Iongitiidinal section. A t this stage. the daughter nnclci are separ;itcd i)y ;I narro\\ diainetw - 0.3 I c i n ) isthniiis i.rgion which niay e z c ~ i ~ t l 2.0 / ,ti11 in Irn,qth.

Throughoiit t h e proems of nuclear elorigation the nu(-leal rnvrlopr rrniainv intact. ;\lthough chromatin is less dirtingiiish- aI)lr duriiy i i i i tos i \ th; t i i a t interphaw, it i.; apparent that some re,qionz o f i t 2rc i n rontz i rr 1% it11 t h r niiclear prt.iphery isre Figs. -!, ti. 8 , I!) , . 11 is tt.tiiptinq to s p c ~ u l a t c that t h c w rryions are

identical tn the prripheral chromatin a t interphase, i.c. that trypanosome c h r o ~ n ~ ~ o n i e ~ h a v c x permanent attachrnent sites at the ririclcnr envelope.

T h e nucleoliis dispeixs early in mitosis but granular material of presitinptive niic.leolar origin is often recognizable in mitotic nuclei where it occnrs closc to the spindle rnicrotnhules. I n late initosis, nucleoli are iefornird in the region traversed by spindlc niicmtul)ulrs. Consequently in electron inirrographs a t these and snl~ceqiirnt stages, the spindle is seen in close association with the nuclrolus (Figs. 17 -19) .

Some mitotic nuclei contain as inany a s 4 electron-densr structiirrs of unkno\vn nature. One of thesc strnctures is indicated in F ip . 6-8 and shown at a highrr niagnification in Figs. 10-12. This structure consists of 2 similar plaques, 125 nm wide and 60 nni thick, separated by a less dense region. A strncture reseinhling one of these plaqnes (140 X GO nrn), seen in another nucleus (Figs. 13-15), may lie a division product of the double plaque. A 2nd type of electron-dense structure in Initotic nuc.lci of T. cyclops is spherical or cylindrical, ranging froin 80 nni in clianietcr to 160 nni in ltmgth. Its relationship to the plaque-like .;triictiires is not known. Although spindle inicrotulinles run closr t o thrse electron-dense striicturrs (Figs. 10-15) I there is no ovi- clence of insertion; howcver, a possihlr lateral connrction is indicated by the small arimvs in Fig. 14.

Replication of thc kinetoplast and the Imal body of the flagdlum occurs Iwfore niitosis, and thesc striicturrs rrtain their i.l<i.;e priixilility to the nucleus during thr process of division.

MITOSIS IN Trypanosoma cyclops 289

Figs. 3-8. Serial sections through a nucleus in early mitosis. Spindle rnicrotubuIes ( s ) and condensed peripheral chromatin (c) are

Fig. 9. Composite line diagram of the spindle shown in Figs. 3-8. Spindle microtubules are approximately parallel to each other and indicated in Figs. 4, 6, and 8. The electron-dense structures (arrows) are shown at higher magnification in Figs. 10-12. x 35,000.

extend from the nuclear periphery a t one pole across the nucleus toward the opposite pole. x 35,000.

290 ~I ITOSIS I N Tiypanosoma cyclops

Tliry do not, holvever, have a morphologic involvement in mito&, e.g. the has;tl bodies do not serve as spindle pole bodies. Spindle microtuliulcs terminate close to the inner surface of the nuclear envelope. No striicture reminiscent of a spindle pole I~ody has heen seen.

DISCUSSION Several details of mitosis in T. cyclops resemble those de-

scrilxd for other species of trypanosomes (2, 4, 10, 17, 19). For example, mitosis is intrannclear, spindle pole bodies are absent, and during nuclear elongation a narrow isthmus forms between thc daughter nuclei. I n common with T. cruzi ( 4 ) , T . raiae i 19) , and T . rhodc.rien.rc ( 19), electron-dense strnctiires occur iii mitotic nuclei h i t thesc are not identical to those reported rai-lirr. For esaniple, the “laiiiinated plaque-like structures” in T . rniac and T. rhodcsicnse are larger than those seen in T. c.yclops, possess a different siihstructure, and appear to be more intiinately associated Ivith spindle microtubules. Although the function of these structures in T . cyclops remains unknown, it chould l ~ e recalled that they are absent in interphase nuclei, and, thcrrforc, it is proliable that they are involved with the mitotic process.

The fate of the nucleolus during trypanosoine mitosis is highly variable-it niay remain intact ( 14) , it may fragment (17, 19), or it may dispcmr (1.). Although this range of behavior is known from other protists ( 7 ) , it is of interest to note its occurrence ivithin a single genus. Material of presuniptive nuclcolar origin can often lie recognized in the vicinity of the spindle micro- tulirilrs. The mitotic spindles in T. rhodesit:nse and T. vivax are similarly associated ui th fragnicnted parts of the nucleolus ( 17, 1 9 ) .

T h e appearance of chroniatin in the nuclei of trypanosoines varies \viddy. In T . c t p i p d u ~ ~ it is dispersed in hoth interphase and niitotir nnclvi (13). In T . cruri (4 ) chromatin can lie recognized in the interphase nucleus as electron-dense material at the nuclear periphery; hoLvevel-, the chromatin disperses dur- ing mitosis and conscquently an association with other nuclear coniporirnts such ax the spindlr or nuclrar envelope cannot be dcinonstrated. In T. cyclops, chromatin is less densely stained during niitosis, Init nevertheless i t is recognizable and can be ol~scrved in contact with the nuclear periphery. An association Iwt\teen mitotic chromatin and the nuclrar periphery has also Ixwi reported in T . vioar ( 1 7 ) and in bloodstream forms of 7‘. rhodc~icnsc ( 19). Tlicse observations led Vickerman & Prrqton to speculate that the nuclear envelope “acts as a carrier in the hipartition of genetic material, the spindle serving to piish apart thc tivo halves of the nuclear envelope” (19 ) . It is well k n o u 11 that a dirwt association I,et\veen chroinosoiiies and thc iiiitotic spindlc is lacking in soiiie dinoflagellates ( 12, 16). Chro- inosoines are attached to th r pc stent nuclcar envelope, which <ecin to lie iiivolved in their segregation into daughter nnclei.

c

Figs. 10-15. [Serial sections. x 80,000.1 10-12. Higher mag- nifications o f the double strurture (arroivs) shown in Figs. 6-8. 13-15. Structures (arrows) resembling one component of the double structure shown in Figs. 10-12. Most of the structure is present in Fig. 14. Only one edge is seen in Fig. 13, while thc nurleoplasni on its opposite side is evident in Fig. 15. A possible lateral connection betlveen the electron-dense structure and the spindle rnicrotuhules is indicated by the small arrows in Fig. 14.

T\vo spindle microtubules surrounded by granular mate- rial of presumably nucleolar origin. Two interniicrotubule bridges are indicated by arrowheads. x 100,000.

Fig. 16.

MITOSIS I N Trypanosoma cyclofis 29 1

Figs. 17-19. Serial sections through part of the cell shown in Fig. 2. Peripheral chromatin ( c ) , a re-forming nucleolus ( n ) , and spindle microtubules ( s ) are present in this nucleus which is a t a late stage in mitosis. Pigment bodies ( p ) are present in the cytoplasm. The isthmus region ( i ) passes out of the plane of section close to the division furrow which is lined by pellicular microtubules (prn). x 50,OOU.

Cross sections through a nucleus in a division stage similar to that shown in Figs. 17-19. The section between Fig. 20 and 21 is not shown; Figs. 21-25 are serial. These sections were cut across the isthmus region (Figs. 20-23) close to the point where it broadens into one of the daughter nuclei (Figs. 24, 25) . X 50,000.

Figs. 20-25.

I t is possible that the nuclear envelopc nf T . C ~ C ~ O / J S is likci\.ise involved in genome separation-ihc attac-iiincnt of chromatin to the nnclear envelope of mitotic nriclci i \ a strong indication that this may be the case. T h e ahs~11ce of kinetochores is also significant, but not concluivc, since i t ic conrciv:iljle that niicro- tiibiiles might tri-niinatc a t a rhroino\onic in a structwe not recognizable as a kinctochore. If chl.oinosoinal iiiici.otuIiules are present, however, the number of inicrotnbules shonld vary along the length of the niitotic spindle according to \\-hcther a given region contains only pole-to-pole riiicrottihriles or both pole-to- pole and chroinosomal microtuhules 13. 15) . This is not the case-composites of niitotir spindles ‘ e.g. Pix. 9) indicate that the numher of microtubirles is approxiiiiatcly constant along the length of the spindle. Hence it appear, that onty pole-to-pole microtuhules are present and that the association of chroniatin with the nuclear envelops is important in its segrpgation to dariahter nuclei. It is irrelr\.ant Ishether niiclcar elongation dur- ing mitosis is primarily due to g r m th of the spindle ( 1 , 5 ) or to growth of the nuclear envelope ( 1 3 ) . Both these proccsses occur during tr)-panosoiiie mitosis, and there is 110 experimental evidence for considering n n r to hc inorc iniportant than the other. Spindle microttilniles often are sufficiently close to one another that intc.raction is possilile; e.g., inteiiiiirrotril)rlle liridges (6) have heen ohservtd both in T . c y c l n / ~ s (Fig. 16) and T . rainc ( 19). Sliding interaction n i a y occtir lwt\\.ecn these niicro- tubules as has tieen postnlatcd in othrr systems (6, 8 ) . At- tempts are now being inadc to ol)tain serial transverse sections across whole mitotic nuclei so that the nuiii1)ers of niicrotubiilcs and their arrangement c3n lie inore precisely described for differ- ent stages of mitosis.

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7. Heywood P, Magee PT. 1976. Meiosis in protists. Some structural and physiological aspects of meiosis in algae, fungi, and protozoa. Bacteriol. Rerj. 40. 190-240.

8. ----, Van De Water TR, Hilding DA, Ruben RJ. 1975. Distribution of niicrotuhuks and microfilaments in developing ves- tibular sensory epithelium of mouse otocysts grown in vitro. 1. Cell Sci . 17, 171-89.

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10. Inoki S, Ozeki Y. 1969. Electron niicroscopic observations on division of kinetoplasts in Trypanosoma gambiense. Biken J. 12, :i 1-4 1 .

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the spindle and chromosome movement in the yeast Saccharomyces cerez’isiae. J. Cell Sci. 22, 219-42.

16. Soyer MO. 1975. Chromosoniic division and organization in some dinoflagellates. BioSjlstems 7, 306-7.

17. Vickerman K. 1973. The mode of attachment of Tryp- nnosouia zjivax in the proboscis of the tsetse fly Clossina fuscipes: an ultrastructural study of the epimastigote stage of the trypano- some. J. Prototool. 20, 394-404.

18. ___ 1974. The ultrastructure of pathogenic flagellates. In Trypanosnniiasir and Leishmaniasis With Special Reference to Chagas’ Disease. Ciba Foundation Sympos ium 20 ( n e w series). Elsevier, Amsterdam, pp. 171-98.

19. ---, Preston TM. 1970. Spindle microtubules in the dividing nuclei of trypanosomes. J. Cell Sci. 6, 365-83.

20. Weinman D. 1972. Trypanosoma cyclops n. sp.: a pig- mented trypanosome from the Malaysian primates Macaca nemes- trina and M. ira. Trans . R . SOC. T T O ~ . M e d . H y g . 66, 628-36.

Rottger, R. ./. Protozool. 25 ( 1 ), 31-44. Page 42, legend for Figure 4 should read:

Fig, 4. Schematic. diagrams of the regeneration sequence in individual No. 2 , Table 1 (duration of regeneration 31 days). The areas CJf the lest which contain protoplasm are dotted. a. February 11: 1970. one day after niultiple fission; there were small amounts of residual protoplasm in some chamberlets of the youngest chamhers. “r faint hue in the oldest part of the test indicates the presence of some residual protoplasm also in this part o f the test. b. February 19, 1970. c . March 5: 1970. d. March 13, 1970. A hole in the youngest chamber \\as repaired.

1978. Unusual multiple fission in the garnont of the larger foraminiferan Heterostegina depressa.