schizogony in Plasmodium falciparum and investigation of ... Key words: Plasmodium falciparum, immunofluorescence, microtubules, ... motility and transport.
Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin M. READ1, T. SHERWIN 2 , S. P. HOLLOWAY1, K. GULL 2 and]. E. HYDE1* 1
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD, UK 2 Department of Biochemistry and Molecular Biology, The Medical School, University of Manchester, Manchester Ml3 9PT, UK (Received 21 May 1992; revised 19 August 1992; accepted 1 September 1992) SUMMARY We describe a novel procedure for the immunofiuorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of atubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum a-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue. Key words: Plasmodium falciparum, immunofluorescence, microtubules, mitosis, post-translational modifications, acetylation.
INTRODUCTION
Recently, cloning of the two a-tubulin and single /?tubulin genes of Plasmodium falciparum has shown that the tubulin molecules of this parasite are highly conserved relative to those of a wide variety of other eukaryotes (Delves et al. 1989; Holloway et al. 1989, 1990), consistent with their ancient and crucial role in numerous processes, including cell division, motility and transport. As yet, native tubulin has not been purified from P. falciparum. However, the high degree of sequence conservation predicted from analysis of the genes led us to ask whether wellcharacterized cross-reacting antibodies originally raised against tubulin molecules of different organisms (Trypanosoma brucei and yeast in this case) could be used as fluorescent probes for microtubular structures in the replicating malaria parasite, as well as for various post-translational modifications known to occur in certain cases. Immunofluorescence is a powerful technique that enables the distribution and * Reprint requests to Dr J. E. Hyde, Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology (UMIST), P.O. Box 88, Manchester M60 1QD, UK.
modulation of selected macromolecular components to be visualized within individual cells in a population. We reasoned that if preparative conditions could be established that maintained the integrity of microtubular fine structure in the parasite, and allowed satisfactory visualization thereof, such conditions could be used in future for examining other macromolecular complexes of interest, as appropriate antibodies are identified or become available. The changes in microtubular organization expected as mitosis proceeds make this a particularly attractive system for study in the malaria parasite. Previous studies of the nuclear divisions of (mainly rodent) Plasmodium species, largely using electron microscopical techniques, have shown these to be distinctive and unlike the classical model derived from higher eukaryotes. They were classified according to the scheme of Hollande (1972) as cryptomitoses, where the nuclear membrane remains intact throughout division. A basic pattern is common to all the divisions occurring in the lifecycles of the Plasmodium species that have been investigated. A single centriolar plaque (kinetic centre) engaged in a pore of the nuclear membrane gives rise to a half spindle (hemispindle). T h e
Parasitology (1993), 106, 223-232 Copyright © 1993 Cambridge University Press 17-2
M. Read and others centriolar plaque subsequently divides, simultaneously splitting the hemispindle to produce two daughter hemispindles. The latter then move into opposition by the migration of the centriolar plaques in the nuclear membrane, to produce the complete spindle. This has been shown in the case of sporogony in P. berghei (Schrevel, Asfaux-Foucher & Bafort, 1977) to consist of three different sets of microtubules; one extending from pole to pole of the spindle, another attached to one pole and extending to a point somewhere beyond the equatorial plane of the spindle, and the third type connecting each kinetochore to one spindle pole. Karyokinesis occurs without the chromatin becoming condensed (Aikawa & Beaudoin, 1968; Canning & Sinden, 1973; Vivier & Vickerman, 1974), although Sinden, Canning & Spain (1976) showed, in marked contrast, that condensation of chromatin occurs in the last of the three mitotic divisions of gametogenesis in P. yoelii. In this work we have used the term nuclear bodies to describe the masses of DAPI-positive nuclear material shown in the images illustrating mitosis. These could be interpreted either as separate nuclei or as discrete aggregations of chromatin within a single nucleus. Microtubule-associated structures have been shown to be important in the post-karyokinetic cytodifferentiation phase of schizogony and sporogony in P. berghei and gametogenesis in P. yoelii (Sinden, 1978). Here, we also describe structures that are associated with the equivalent late phase in the erythrocytic schizogony of P. falciparum. Development of the immurtofluorescence technique described here, which has allowed the visualization of the malarial parasite microtubule organization in the different stages, represents a significant improvement on current protocols, and should prove to be of general applicability. As well as using a general anti-a-tubulin antibody to examine microtubular structures, we tested by immunofluorescence microscopy and immunoblotting whether two further monoclonal antibodies specific for important post-translational modifications of a-tubulin would bind to P. falciparum molecules. In virtually all sequenced a-tubulins, the terminal encoded amino acid is Tyr. However, a specific tubulin tyrosine carboxypeptidase (TTC) has been described which acts preferentially on the a-tubulin of assembled microtubules (Kumar & Flavin, 1981). This catalyses the progressive loss of the Tyr residue as microtubules age, exposing the penultimate C-terminal residue, which is normally Glu. After microtubule disassembly, a tubulin tyrosine ligase (TTL) can add a new Tyr residue to this Glu residue, a step that may have significance in the recycling of components (reviewed by Cleveland & Sullivan (1985) and Burns (1987)). In the case of P. falciparum a-tubulin I, which is constitutively expressed (Delves et al. 1990), a terminal Tyr is also
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encoded in the DNA sequence (Holloway et al. 1989), although the molecule is unusual in that it ends in Asp-Tyr, rather than Glu-Tyr. A further important modification centres on the
lysine residue at position 40 of most a-tubulins. This residue is subject to a highly specific post-translational acetylation, first identified in Chlamydomonas (LeDizet & Piperno, 1987) and now characterized in a number of organisms (MacRae & Langdon, 1989). A corresponding lysine residue is present in a-tubulin I of P. falciparum, but not in the sexual stage-specific a-tubulin II molecule, where position 40 is a glutamine residue (Holloway et al. 1990). Our results also shed light on the status of these modifications in the erythrocytic stages of P. falciparum. MATERIALS AND METHODS
Parasites, isolate Kl (Thailand), were cultured in 25 cm2 tissue-culture flasks as previously described (Read & Hyde, 1988). The antibodies TAT1 (a-tubulin specific) and C3B9 (acetylated a-tubulin specific) were both raised against T. brucei in one of our laboratories (Woods et al. 1989). YL1/2 (tyrosinated a-tubulin specific, originally raised against yeast) was the kind gift of John Kilmartin (Kilmartin, Wright & Milstein, 1982). Synchronization of parasite cultures Four parallel synchronous cultures were produced using sorbitol treatment, which is preferentially lethal to late trophozoites and schizonts (Lambros & Vanderberg, 1979). Parasitized blood at 10% parasitaemia was exposed to a 5 % sorbitol solution (in phosphate-buffered saline, PBS) for 5 min at room temperature. The sorbitol was then removed by washing the blood in complete medium. The parasitized blood, supplemented with an equal volume of uninfected blood, was then returned to a culture flask with medium to a haematocrit of 5%. To sharpen the synchrony, this procedure was repeated one cell cycle (i.e. 48 h) later. The length of the period between establishing new ring-stage parasites and onset of the next round of schizogony is approximately 33 h (Hall et al. 1984; Inselburg & Banyal, 1984). Following the second sorbitol treatment, we therefore allowed 315 h to elapse before taking samples for immunofluorescence assay; sampling was then carried out at hourly intervals over the following 12 h. Over this period, during which the parasites were healthy and developing normally, the parasitaemia was 5-6%. Preparation of P. falciparum for immunofluorescence At each time-point 15 /tl of blood was taken from the red blood cell layer on the floor of each culture flask.
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Immunofluorescence of P. falciparum microtubules
The blood from the 4 flasks was combined into 1 microfuge tube. This volume produced enough material for 2 slides. An equal volume of 3-7 % formaldehyde (in PBS) was then added and gently mixed in using a pipette. The samples were then stored in this state at 4 °C overnight. Next day each of the 12 samples was diluted a further 5-fold in 37 % formaldehyde. Poly-L-lysine-treated slides were prepared by immersion in a 100/
