J. Euk. Microhiol., 46(2), 1999 pp. 105-109 0 1999 by the Society of Protozoologists
Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle LINDA KOHL,*,’ TREVOR SHERWIN* and KEITH GULL* *School of Biological Sciences, University of Manchester, Manchester M I 3 9PT, United Kingdom
ABSTRACT. Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On Western blot, they detect a doublet of high molecular weight (M, 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates. Supplementary key words. Monoclonal antibodies, trypanosomes.
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HE African trypanosome Trypanosom brucei is responsible for sleeping sickness in humans and a similar disease in cattle. The parasite undergoes a complicated life cycle, alternating between an insect vector and a mammalian host. The shape and form of the T. brucei cell is maintained by an internal cytoskeleton: a corset of subpellicular microtubules that are present at all stages of the cell cycle [I, for a recent review: 71. Trypanosoma brucei possess a single flagellum that is attached to the cell body via a complex flagellum attachment zone (FAZ) [12]. The FAZ is defined as comprising two different structures: a unique cytoplasmic filament complex and four specific microtubules situated next to it. Lateral projections from the cytoplasmic FAZ filament connect to the plasma membrane and then this membrane and the flagellar membrane are adpressed to each other. Within the flagellum, filaments link from this position to the paraflagellar rod (PFR), a unique, latticelike structure present alongside the axoneme [2, 121. It has been shown recently that the PFR plays an important role in cell motility [3]. We know the identity of the major PFR proteins, such as PFR-A and PFR-C gene products [2, 4, 111. However, we have very little knowledge of the proteins comprising the FAZ structure. One of the most obvious features of the trypanosome cell cycle is the elaboration of a new flagellum. In procyclic T. brucei, the initial stage of flagellum growth involves the duplication of the basal bodies at around 0.41 of the unit cell cycle [12, 151. The new axoneme then elongates and, when it emerges from the flagellar pocket, growth of the new paraflagellar rod is observed at 0.52 of the unit cell cycle. Flagellum growth then continues through much of the cycle [9, 121. In order to identify new components of both complexes, and to provide probes to address questions of coordination of assembly of these structures during the cell cycle, we have raised a set of new monoclonal antibodies against the FAZ and the PFR from T. brucei. The FAZ complex is important not only for flagellum attachment, but it also seems likely to be implicated in cytokinesis [9]. Therefore, it is essential to know how the growth of these structures (the external flagellumPFR and the cytoplasmic FAZ) are orchestrated during the cell cycle. Three main models can be imagined: 1) both structures are elaborated in a coordinated manner [9]; 2) the external PFR starts growing before the internal FAZ; 3) the internal FAZ starts growing first and the growth of the PFR follows as a second step. In the last two situations, the elongation rate of the internal FAZ and external
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PFR has to be determined to know whether one complex grows faster than the other. Here, using the new monoclonal antibodies, we have shown for the first time that during the cell cycle, growth of the internal FAZ filament is initiated before the external, attached portion of the flagellum, as monitored by the appearance of the new PFR. Moreover, we show that the two structures, once initiated, do not grow at the same rate. MATERIALS AND METHODS Partial purification of the flagellum-associatedstructures. Procyclic trypanosomes (5 X lo8 cells) were harvested by centrifugation (1,000 g at room temperature for 5 min), washed once in phosphate-buffered saline (PBS: 137 mM NaCl, 3 mM KC1, 7 mM Na,HPO,, 1 mM KH,PO,, pH 7.4), and collected by centrifugation. The cell pellet was resuspended in 1% Nonidet P40 in 0.1 M Pipes, pH 6.9, 2 mM EGTA, 1 mM MgSO, and incubated for 3 min at room temperature to provide a cytoskeleton preparation [lo, 121. After centrifugation (1,000 g at room temperature for 5 min), the pellet was resuspended in 60 mM CaC1, to depolymerise the subpellicular microtubules, incubated for 15 min at room temperature, and centrifuged. The pellet was resuspended in PBS. This preparation, containing the axoneme, the PFR, the FAZ filament, and the nuclear remnants, was called “Ca2+ flagellar preparation.” Production of monoclonal antibodies. Three mice (footpad injection) were injected with the “Ca2+ flagellar preparation” emulsified in complete Freund adjuvant. After 10 d the mice received a boost with the “Ca2+ flagellar preparation” (same concentration as above) in PBS. The mice were sacrificed 4 d later. Their lymph nodes were removed and used for fusion with myeloma cells X63-Ag8.653 [8]. After 10 d at 37” C, the hybridoma supernatants were tested by immunofluorescence on methanol-fixed trypanosomes [ 131. Four antibodies were selected for further subcloning and analysis. Immunoelectron microscopy and immunofluorescence. Imrnunoelectron microscopy and immunofluorescence were performed on whole cells and detergent-extracted cytoskeletons as described by Sherwin and Gull [12, 131. Immunofluorescence images of 56 methanol-fixed trypanosomes and 97 detergent-extracted cytoskeletons were captured on a slow scan CCD camera (Photometrics CH 250, USA) and used for measurements of new FAZ and new PFR (application IPLab Spectrum [Image analysis software, Signal Analytics, USA]). The length of new FAZ was plotted as a function of the length of new PFR, using Cricket Graph (Computer Associates International, USA) and the equation for the linear relation was determined. Western blot. Western blot analysis was performed on de-
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Fig. 1. Biochemical and structural characterisation of the monoclonal antibodies to the flagellum attachment zone (FAZ) and paraflagellar rod (PFR)of Ttypano.soma brucei. a. Immunofluorescence using antibodies L8C4, L13D6, L6B3 and L3B2 on whole trypanosomes. Merged phase
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tergent-extracted cytoskeletons as described by Sherwin et al. [ 131, incorporating 0.1 % sodium dodecyl sulphate for the transfer of high molecular weight proteins. Detection was realised by chemiluminescence, using the ECL kit (Amersham, Little Chalfont, Buckinghamshire, UK). RESULTS We have partially purified the flagellum-associated structures of T. brucei by combining detergent-extraction [ 121 and treatment with 60 mM CaC1,. The calcium treatment depolymerised all the subpellicular microtubules, leaving only the axoneme, the PFR, the FAZ filament, and the nuclear remnants. This preparation, called “Ca2+ flagellar preparation”, was injected into mice. Immunofluorescence was used to screen the hybridoma supernatants for interesting antibodies. Four antibodies were selected, recognising either the PFR (L8C4 and L13D6) or the FAZ (L3B2 and L6B3). The pattern of staining, using immunofluorescence on methanol-fixed trypanosomes, revealed that the anti-PFR antibodies L8C4 and L13D6 exclusively detected the flagellum from its exit from the flagellar pocket to the flagellar tip (Fig. la). The staining pattern of the anti-FAZ antibodies L6B3 and L3B2 followed the region between the cell body and the flagellum from the flagellar pocket to the anterior end of the cell, but did not extend to the end of the flagellum. Although both anti-PFR antibodies (L8C4 and L13D6) recognised the same structure in immunofluorescence, their pattern on Western blot of detergent-extracted cytoskeletons of T. brucei was different (Fig. lb). First, L13D6 recognised a doublet of M, 69,000 and 73,000, corresponding to PFR-A and PFR-C, as does the monoclonal antibody 2E10, raised previously against PFR proteins from Leishmania [6]. However, the antibody L8C4 recognised only the PFR-A band and not the PFRC band. Both anti-FAZ antibodies (L6B3 and L3B2) recognised a doublet of high molecular weight (M, > 200,000). All the antibodies were tested by immunoelectron microscopy on detergent-extracted cytoskeletons of T. brucei. In these preparations, when the membranes were removed, the flagellum often became partially separated from the cell body, thus allowing easier visualisation of the FAZ. Both anti-PFR antibodies labelled the paraflagellar rod (data not shown). Only the cytoplasmic FAZ filament stained with both anti-FAZ antibodies (Fig. lc). No staining is observed on the PFR, the axoneme or the microtubules. The anti-FAZ antibodies belong to different subclasses: L3B2 is an IgG1, whereas L6B3 is an IgM, therefore allowing easy, clean double labelling. Both anti-PFR antibodies are of IgGl subclass. We used the combination of the anti-FAZ antibody L6B3 with the anti-PFR antibody L8C4, to analyse the kinetics of assembly of these two structures through the cell cycle. Growth of the cytoplasmic FAZ filament and the external PFR did not start at the same time of the cell cycle (Fig. 2). The position of the cell in the cell cycle was indicated by the duplication of the kinetoplast and the nucleus (monitored by DAPI staining) [7, 12, 151, as well as by the state of the basal body, as monitored by staining with the anti-basal body antibody BBA4 [14, 151. Both L6B3 and BBA4 are of IgM subclass and were detected using the same secondary anti-mouse antibody,
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resulting in the labelling of both FAZ and basal bodies (BB) in the same channel (Fig. 2, Panel A). As the cell progressed through the cell cycle, the kinetoplast elongated, the basal bodies duplicate, and a second flagellum (seen in the phase contrast image, indicated by the white arrow) and a second FAZ (indicated by the green arrow) were distinguished (Fig. 2, panel B). However, no second PFR was observed at this stage. At the next step, while the daughter FAZ elongated, a second PFR (indicated by the red arrow) was detected (Fig. 2, panel C). The FAZ filament is anchored very close to the basal body area (Fig. 2). The PFR appears only after exit of the flagellum from the flagellar pocket, as has been shown previously [ 5 ] . These observations were confirmed by measurements of the length of the new FAZ and PFR using both fixed whole cells (n = 56) and detergent-extracted cytoskeletons (n = 97, Fig. 3); both produced the same results. There is a clear lag period between the initiation of the growth of the PFR and that of the FAZ: only when the new FAZ reached a length of about 1.4 pm, did the new PFR start elongating (Fig. 3). As both structures elongate, the FAZ extended more slowly than the PFR since the slope was less than 1.0 (Fig. 3). Thus, the PFR, starting its growth after the FAZ, apparently has a faster growth rate than the FAZ. DISCUSSION We have produced a set of novel monoclonal antibodies against the PFR and FAZ proteins of T. brucei. This is the first description of monoclonal antibodies raised against the PFR in T. brucei, including one (L8C4) that recognises exclusively PFR-A. The sequence of the PFR-A and -C proteins is known and they are very similar (60% amino acid identity throughout). Divergence occurs mostly in three regions: in the amino- and the carboxy-terminal domains and in an internal location (amino acid 524 to 552, numbering of PFR-A [ 2 ] ) . Using truncated versions of the PFR-A, we have excluded the amino- and carboxy-termini of the protein as the L8C4 epitope, leaving the internal sequence as a likely candidate for the difference in antibody recognition site (Bastin, P & K. G., unpubl. data). The novel anti-FAZ antibodies recognise the FAZ filament by immunofluorescence and immunoelectron microscopy. It is still impossible to say whether they recognise the same protein(s), although they detect a similar doublet of high molecular weight (M, 3 200,000). We showed that the FAZ filament originates close to the basal body area. It has been previously described that the 4 microtubules, which are part of the FAZ complex, also initiate very close to the basal body area [ I]. The set of new monoclonal antibodies also allowed us to address the question of the relationship between assembly of the cytoplasmic FAZ and the external flagellum during the cell cycle. During the cell cycle, the assembly of the FAZ structure is initiated before that of the PFR and the two structures elongate at a different rate: the PFR growing at a slightly faster rate than the FAZ. The most obvious overview therefore is that in the cell cycle, growth of the new FAZ filament occurs in the period between 0.4 and 0.5 of the cell cycle unit, around the time of probasal body maturation and new axoneme extension. The new axoneme of the new flagellum then exits from the flagellum
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contrast picture with fluorescence (in white). b. Immunoblot of T. brucei cytoskeletal proteins using anti-FAZ antibodies L6B3 and L3B2 or antiPFR antibodies 2E10, LI 3D6 and L8C4. c. Immunoelectron microscopy of detergent-extracted cytoskeletons of T. brucei using the anti-FA2 antibodies L6B3 and L3B2. For clarity, we have chosen to show areas where the flagellum has come away from the subpellicular corset to reveal the labelling of the FA2 filament. The arrows point towards the FA2 filament where gold particles are localized. Bars = 0.1 Fm.
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ments and discussions, and Dr. D. McMahon-Pratt for kindly providing the antibody 2E10. L.K. has been a recipient of an International Fellowship from the Wellcome Trust. Work in K.G’s lab is supported by a Wellcome Trust Programme Grant and Equipment Grant and a BBSRC Project Grant.
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LITERATURE CITED
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Length of new PFR (pm) Fig. 3. Length of the new flagellar attachment zone (FAZ) of Trypanosoma brucei as a function of the length of the new paraflagellar rod (PFR) measured on 97 detergent-extracted cytoskeletons of dividing cells. Equation of line y = 0 . 6 1 9 ~+ 1.388; r2 = 0.923 where m and b are constants, m defines the slope and b defines the intersection with the y axis.
pocket and PFR growth is initiated. The axoneme extends as does the PFR, but it appears that inside the cell, the FAZ filament extension lags, suggesting that a completed PWaxonema1 structure is required before cross-linking this to the internal FAZ. In electron microscopy, dense connections crosslink the PFR and axoneme, involving the microtubule doublets 4 and 7, and there are connections between the PFR and the internal face of the flagellar membrane, directly opposite of the FAZ filament [12]. This knowledge is important since we have hypothesised that the assembly of the FAZ complex inside the dividing trypanosome may be a critical event in determining the axis and position of cytokinesis [9]. This new set of monoclonal antibodies, particularly monoclonals to the FAZ filament, will allow the molecular identification of some of the FAZ components and ultimately permit testing by molecular genetics of our hypothesis that FAZ is involved in cytokinesis. ACKNOWLEDGMENTS We thank Dr. K. Ersfeld for excellent assistance in the raising of the monoclonal antibodies, Dr. €? Bastin for helpful com-
1. Angelopoulos, E. 1969. Pellicular microtubules in the family Trypanosomatidae. J. Protozool., 17:39-5 1. 2. Bastin, l?, Matthews, K. R. & Gull, K. 1996. The paraflagellar rod of Kinetoplastida: solved and unsolved questions. Parasirol. Today, 12: 302-307. 3. Bastin, l?, Sherwin, T. & Gull, K. 1998. Paraflagellar rod is vital for trypanosome motility. Nature, 391548. 4. Deflorin, J., Rudolf. M. & Seebeck, T. 1994. The major components of the paraflagellar rod of Trypanosoma brucei are two similar, but distinct proteins which are encoded by two different gene loci. J. Biol. Chem., 269:28745-2875 1. 5 . Fuge, H. 1969. Electron microscopic studies of the intra-flagellar structures of trypanosomes. J. Prorozool., 16460-466. 6. Ismach, R., Cianci, C. M. L., Caulfield, J . €?,Langer, €? J., Hein, A. & McMahon-Pratt, D. 1989. Flagellar membrane and paraxial rod proteins of Leishmania characterisation employing monoclonal antibodies. J. Protozool., 36:617-624. 7. Kohl, L. & Gull, K. 1998. Molecular architecture of the cytoskeleton. Mol. Biochem. Parasitol., 93: 1-9. 8. Lignau, A., Chakrabony, T., Niebuhr, K., Domann, E. & Wehland, J. 1996. Identification and purification of novel internalin-related proteins in Listeria rnonocytogenes and Listeria ivanovii. Infect. Immun., 64:1002-1006. 9. Robinson, D. R., Sherwin, T., Ploupidou, A., Byard, E. H. & Gull, K. 1995. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J. Cell. Biol., 128:1163-1172. 10. Robinson, D., Beattie, I?, Sherwin, T. & Gull, K. 1990. Microtubules, tubulin, and microtubule-associated proteins of trypanosomes. Methods Enzymol., 196:285-299. 11. Schlaeppi, K., Deflorin, J. & Seebeck, T. 1989. The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes. J. Cell. Biol., 109: 1695-1709. 12. Sherwin, T. & Gull, K. 1989. The cell cycle division of Trypanosoma brucei brucei: timing of event markers and cytoskeletal modulations. Phil. Trans. R. Soc. Lond., B323:573-588. 13. Sherwin, T., Schneider, A,, Sasse, R., Seebeck, T. & Gull, K. 1987. Distinct localisation and cell cycle dependence of COOH-terminally tyrosinated a-tubulin in the microtubules of Trypanosoma brucei. J. Cell. Biol., 104:439-446. 14. Woods, A., Sherwin, T., Sasse, R., MacRae, T. H., Baines, A. J. & Gull, K. 1989. Definition of individual components of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci., 93:491-500. 15. Woodward, R. & Gull, K. 1990. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J. Cell Sci., 9549-57. Received 10-6-98, 2-1-99; accepted 2-2-99
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Fig. 2. Immunofluorescence of the detergent-extracted cytoskeleton of Trypanosoma brucei during the cell cycle (A-D). Cells were doublelabelled with the anti-paraflagellar rod (PFR) antibody L8C4 (shown in red) and the anti-flagellar attachment zone (FAZ) antibody L6B3 (shown in green). The position of the cell in the cell cycle is indicated by the DNA staining dye 4,6-diamidino-2-phenylindole(Dapi, shown in bluewhite) where Panel A is in the GI stage and Panel D has completed mitosis. The new flagellum is indicated by a white arrow in phase images; the new PFR by a red arrow in Anti-PFR images; and the new FAZ by a green arrow in the Anti-FAZ/Anti-BB images. The rightmost panels show merged images of Anti-PFR and Anti-FAZIAnti-BB images. magnified 200%. BB = basal body; K = kinetoplast; N = nucleus.
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