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Dec 31, 1996 - PROTISTOLOGY. The Ultrastructure of Mitosis in the Free-Living. Kinetoplastid Bodo curvifilus. AlexanderO. Frolov*, Serguei A. Karpov**, and.
Europ. J. Protisto!' 32, 498-505 (1996) December 31, 1996

European Journal of

PROTISTOLOGY

The Ultrastructure of Mitosis in the Free-Living Kinetoplastid Bodo curvifilus AlexanderO. Frolov*, Serguei A. Karpov**, and Marina N. Malysheva* *Laboratory of Protozoology, Zoological Institute of Russian Academy of Sciences, St. Petersburg, Russia **Biological Research Institute of St. Petersburg State University, Russia

SUMMARY The nuclear division of a free-living bodonid flagellate was investigated at the ultrastructurallevel for the first time. Transmission electron microscopy of serial ultrathin sections shows that nuclear division of Bodo curvifilus occurs by closed mitosis with an intranuclear spindle, as in trypanosomatids and cryptobiids. The nucleolar material of B. curvifilus persists during the nuclear division and seems to be shared between the daughter nuclei. The spindle consists of one compact bundle of approximately 30 microtubules running through the nuclear centre: most of the microtubules continue from one pole to another. Microtubule-organising centres of the mitotic spindle lie at the opposite poles, and they are not distinguished morphologically. The spindle of B. curvifilus is associated with at least 10 pairs of kinetochore-like dense plaques. In sections only the plaques can be seen at different stages of migration towards the poles as chromosomes are not condensed during mitosis of kinetoplastids.

Abbreviations CH CP CMT F FP FV KP M MT N NU P

=

chromatin

=

flagellum

= cytopharynx = cytopharyngeal microtubules = flagellar pocket = food vacuole = kinetoplast

= = =

mitochondrion spindle microtubules nucleus = nucleolus = kinetochore-like dense plaques

Introduction There are three families of kinetoplastid flagellates: free-living biflagellate bodonids, predominantly parasitic biflagellate cryptobiids, and obligatory parasitic uniflagellate trypanosomatids. 0932-4739-96-0032-0498$3.50-0

The ultrastructure of the mitotic nucleus in trypanosomes is well known [6, 7, 9, 10, 13, 14, 15] and represents a closed intranuclear mitosis without chromosomal condensation. In cryptobiids the nuclear division has been investigated in the only species Cryptobia (= Trypanoplasma) borreli - a parasite of fish blood [5] and is similar to that of trypanosomatids. The mode of nuclear division in free-living bodonids is not yet known, and so we have an incomplete picture of mitosis for the Kinetoplastida as a whole [16]. In this paper, we present the ultrastructure of the mitotic nucleus in the bodonid Bodo curvifilus.

Material and Methods The strain of Bodo curvifilus was isolated with accompanying bacteria from a sample collected during an expedition on the program "Marine Ecological Patrol '94" around the Baltic Sea in August 1994. The strain was adapted to the fresh-water habitat, and cultivated on Pratt media 0.1 gil © 1996 by Gustav Fischer Verlag, Stuttgart

Mitosis in Bodo curvifilus . 499

Figs. 1-4. Ultrathin sections of cell (1) and nucleus (2 -4) of Bodo curvifilus. - Fig. 1. Position of the nucleus in the flagellate. Figs. 2, 3. Fine structure of the interphase nucleus. - Fig. 4. Fine structure of the nucleus at the early prophase. Scale bar in Fig. 1 = 1 ~m, Figs. 2-4 = 0.5 ~m.

KN0 3, 0.01 gil MgS0 4·7H ZO, 0.01 gil KzHP0 4·3H zO, 0.001 gil FeCI 3·6H zO, pH 7.0) enriched by bacteria Aerobac-

ter (= Klebsiella) aerogenes.

To obtain a synchronously dividing culture, the strain of

B. curvifilus was kept at room temperature for 7 days, then

transferred into fresh medium. The cells began to divide quite synchronously, and were fixed for the EM during the exponential phase of growth. B. curvifilus was collected by centrifugation and fixed with cold mixture (1.5% glutaraldehyde

and 1.5% OS04 in 0.1 M cacodylate buffer, pH 7.2) for 30 min. Postfixation was carried out with 2 % OS04 in 0.1 M cacodylate buffer, pH 7.2, for 1 h. After dehydration in an ethanol series and infiltration with propylene oxide, the material was embedded in an epon-araldite mixture. Ultrathin sections were stained with saturated aqueous uranyl acetate (1 h) and lead citrate (5 min). The sections were examined in a JEM-IOO C electron microscope.

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Mitosis in Bodo curvifilus . 501 ... Figs. 5 - 6. Ultrastructure of the nucleus of Bodo curvifilus at the metaphase stage. - Fig. 5 Four kinetochore-like dense plaques are located at the equatorial region of the nucleus. Scale bar = 0.5 11m. - Fig. 6. The kinetochore-like dense plaques are associated with microtubules of the spindle. Bars show the layered structure of the plaque. Scale bar = 0.1 11m. - Figs. 7-10. Series of sections of Bodo curvifilus at early anaphase. Of 17 serial sections four electron micrographs are illustrated. Figs. 7-10 represent sections Nos. 5 -7, 9, respectively. The ten dense plaques (numbered) are evident within the nucleus. Scale bar for Figs. 7-10 = 0.3 11m.

Results 1. Interphase

The interphase nucleus of B. curvifilus has a spherical form and lies in the anterior part of the cell body close to the large kinetoplast (Fig. 1). The single nucleolus is slightly acentric (Figs. 1,2). The main heterochromatin is seen as lumps located at the nuclear periphery applying to the inner nuclear membrane. Some separate masses and threads of heterochromatin are spread through the karyoplasm, which has a heterogeneous structure. In some interphase nuclei the main mass of heterochromatin can be seen mainly in the karyoplasm as thick threads with a bead-like structure (Fig. 3) and here is little heterochromatin attached to the nuclear membrane in this kind of nucleus. 2. Prophase

In micrographs we recognize the nucleus of B. curvifilus as being in early prophase if there are neither condensed heterochromatin nor microtubules visible (Fig. 4). The karyoplasm of this type of nucleus is more homogeneous than in interphase nuclei. In such micrographs the nucleolus decreases in density and becomes larger. At late prophase the microtubular spindle appears. 3. Metaphase and Anaphase

The microtubular spindle has a prominnent bipolar orientation (Figs. 5 -17) but microtubule organizing centres (MTOC) are not visible at the spindle poles (Figs. 11-17). The microtubules attach directly to the inner nuclear membrane (Figs. 13-15). The spindle is formed by about 30-35 microtubules (Figs. 5, 7 -10). Ten dense plaques appear at the equatorial plane of the dividing nucleus (Figs. 5 -10). They attach to the spindle microtubules and have a layered struc-

ture (Fig. 6) characteristic to the kinetochores of other kinetoplastids [7, 8]. Therefore we can call them kinetochore-like structures. At the beginning of anaphase the nucleus changes its shape from spherical to ellipsoidal (Fig. 11). The spindle elongates and most of the microtubules lie between the poles of the ellipsoidal nucleus (Fig. 11). Kinetochore-like half-plaque movement towards the poles can be inferred from the micrographs represented in Figs. 11-17, which show some dense half-plaques (out of a total of 10) in selected serial sections of the same spindle at anaphase. Thus plaques numbers 1, 3 -7,9 and 10 appear to be "fast moving" in that they are close to the pole while 2 and 8 are "slow" halfplaques lagging behind in a more central position. The fragmented nucleolar material is located around the central part of the spindle (Fig. 11). On the other hand, separate fragments can also be found around the nuclear poles (Fig. 17). 4. Telophase

Further nuclear elongation leads to the typical constricting dumb bell shape (Figs. 18 - 20). In sections of telophase nuclei kinetochore-like structures are absent. The central part of the spindle persits for some time in the channel connecting the daughter nuclei (Figs. 1820) and in completely separated daughter nuclei (Fig. 21). The reconstitution of a dense discrete nucleolus and the beginning of chromatin condensation as peripheral masses occur in both daughter nuclei (Fig. 21). Their full separation marks the end of mitosis in B. curvifilus (Fig. 21).

Discussion The results demonstrate that the nuclear division of B. curvifilus is characterized by intranuclear close mi-

Figs. 11-17. Series of longitudinal sections of Bodo curvifilus in anaphase. Of 12 serial sections seven electron micrographs ~ are illustrated. Figs. 11-17 represent sections Nos. 8, 3 -7, and 9, respectively. The ten dense half-plaques (numbered) are evident within the nucleus. Scale bar for Fig. 11 = 0.5 11m, for Figs. 12-17, scale bar = 0.2 11m. Figs. 18-21. Telophase stage of dividing Bodo curvifilus. - Figs. 18-20. Series of sections at the telophase of mitosis. These serial sections show the thin bridge (arrows) joining the daughter nuclei. - Fig. 21. Daughter nuclei. Scale bar for all figures = 0.5 11m.

~

504 . A. O. Frolov, S. A. Karpov, and M. N. Malysheva

tosis without discernible condensed chromosomes. The organization of the mitotic spindle of B. curvifilus is more similar to that of the trypanosomatids Leishmania [13] and Endotrypanum (Frolov, unpublished data). The microtubules in the spindles of these hemoflagellates form one compact bundle running through the nuclear centre, with the majority of microtubules extending from one pole to the other. The spindle of Leishmania consists of about 60 microtubules, the spindle of Endotrypanum has about 40, and in B. curvifilus there are approximately 30-35 microtubuIes in the spindle. On the other hand, the spindle microtubules of Leishmania and Endotrypanum are associated with 6 pairs of kinetochore-like dense plaques, and the spindle of B. curvifilus has at least 10 pairs. The same number of kinetochore-like dense plaques has been found by Solari in the dividing nuclei of Trypanosoma cruzi [7], but the latter has a spindle of up to 120 microtubules organized in several bundles. There are quite similar mitotic spindles in the nuclei of Cryptobia borreli [5]. Unfortunately, the kinetochore number of C. borreli is unknown. The majority of so called lower trypanosomatids insect parasites - have 20-30 microtubules in their mitotic spindles associated with 3 -4 kinetochore pairs [10, 11]. The nucleolar behaviour may be different during the mitosis of different kinetoplastid species. The nucleolar material of B. curvifilus persists during nuclear division and seems to be shared between the daughter nuclei. A similar pattern of nucleolar behaviour can be seen in Leishmania, Endotrypanum and most "lower" trypanosomatids [9, 10, 11]. Anyway, the nucleolus of T. cruzi disappears in prophase, and the nucleoli are formed de novo in the daughter nuclei [7]. Presented data show that the nuclear division in the free-living kinetoplastid flagellate B. curvifilus belongs to the type of closed intranuclear mitosis. It is very similar to that of parasitic kinetoplastid flagellates-trypanosomatids and cryptobiids [5, 7, 8, 10, 13, 14]. Thus, we can be sure that this type of mitosis is a character of the kinetoplastids as a whole. The main peculiarities of the kinetoplastid mitosis are: a. The nuclear envelope remains intact during the whole process of division. b. Chromatin decondensation occurs at the early stages of mitosis. c. MTOCs of the mitotic spindle lie at the opposite poles and cannot be characterised morphologically. d. There is no chromosome condensation during mitosis but heterochromatin masses are visible in the interphase nucleus. e. Only the kinetochore-like plaques associated with spindle microtubules can be seen to migrate to the opposite poles of the dividing nucleus. This type of mitosis differs essentially from that of euglenoid flagellates. The latter are closely related to the kinetoplastids and both groups obviously represent a monophyletic taxon [1, 2, 3]. Euglenids also have a closed type of mitosis (see [12] for review),

but during the nuclear division of all investigated euglenids the segregation of only condensed chromosomes occurs, and these persist in the interphase nucleus. In addition, there are several microtubular spindles in the dividing nuclei of euglenids.

Acknowledgements We are very grateful to K. Vickerman and anonymous referees for their comments and corrections. The research described in this publication was made possible in part by Grant No. R6R300 from the International Science Foundation and Russian Government.

References 1 Cavalier-Smith T. (1993): Kingdom Protozoa and its 18 phyla. Microbiol. Reviews, 57, 953-994. 2 Karpov S. A. (1990): System of protists. Mezhvuzovskaia typographia, Omsk (in Russian). 3 Kivic P. A. and Walne P. L. (1984): An evaluation of a possible phylogenetic relationship between the Euglenophyta and Kinetoplastida. Origins of Life, 13, 269-288. 4 Raikov I. B. (1982): The protozoan nucleus. Morphology and evolution. Cell BioI. Monogr., 9, Wien, New York. 5 Skarlato S. O. (1987): Fine-structure peculiarities of the nucleus of the parasitic flagellate Trypanoplasma borreli (Kinetoplastida) during interphase and mitosis. Doklady Akademii Nauk CCCR, 293, 220-221. 6 Skarlato S. 0., Lorn J., and Nohynkova E. (1987): Fine structural morphology of the nucleus of Trypanosoma danilewskyi (Kinetoplastida, Trypanosomatina) during mitosis. Arch. Protistenk., 133, 3-14. 7 Solari A. J. (1980a): The 3-dimensional fine structure of the mitotic spindle in Trypanosoma cruzi. Chromosoma, 73, 239-255. 8 Solari A. J. (1980b): Function of the dense plaques during mitosis in Trypanosoma cruzi. Exp. Cell Res., 127, 457460. 9 Solari A. J. (1982): Nuclear ultrastructure during mitosis in Crithidia fasciculata and Trypanosoma brucei. J. Protozool., 29, 330-331. 10 Solari A. J. (1983): The ultrastructure of mitotic nuclei of Blastocrithidia triatomae. Z. Parasitenkd., 69, 3-15. 11 Solari A. J. and De Souza W. (1983): Presence and comparative behavior of mitotic plaques in five species of Trypanosomatidae. Microsc. Electr. Biolog. Cellular, 7,2843. 12 Triemer R. and Farmer M. (1991): The ultrastructural organization of the heterotrophic euglenids and its evolutionary implications. In: Patterson D. J. and Larsen J. (eds.): The biology of free-living heterotrophic flagellates. The Systematics Association special volume, 45, pp. 185 -204. Clarendon Press, Oxford. 13 Urena F. (1986): Three-dimensional reconstructions of the mitotic spindle and dense plaques in three species of Leishmania. Z. Parasitenkd., 72, 299-306. 14 Vickerman K. and Preston T. M. (1970): Spindle microtubules in the dividing nuclei of trypanosomes. J. Cell Sci., 6,365-383.

Mitosis in Bodo curvifilus . 505 15 Vickerman K. and Preston T. M. (1976): Comparative cell biology of the kinetoplastid flagellates. In: Lumsden W. H. R. and Evans D. A. (eds.): Biology of the Kinetoplastida, 1, pp. 35 -130. Academic Press, London.

16 Vickerman K. (1991): Organization of the bodonid flagellates. In: Patterson D. J. and Larsen]. (eds.): The biology of free-living heterotrophic flagellates. The Systematics Association special volume. 45, pp. 15 -184.

Key words: Kinetoplastida - Bodonidae - Nucleus - Nuclear division - Mitosis Alexander O. Frolov, Laboratory of Protozoology, Zoological Institute of Russian Academy of Sciences, Universitetskaya 1, St. Petersburg, 199034, Russia