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preequilibrated with C buffer. The tubulin will bind to the DEAE. Wash the column with 200 ml of C buffer containing 0.35 M NaCI and then elute the tubulin from the column with C buffer containing 0.65 M NaCI. Add 0.38 g/ml ammonium sulfate to the tubulin containing fractions and mix gently. Leave on ice for 20 rain and centrifuge at 27,000 g for 20 min at 0 ° to precipitate the tubulin. Resuspend the pellet to a concentration of 4.0 mg/ml in PEM buffer containing 0.1 m M GTP and 2.0 m M DTE and desalt the tubulin on a spin column containing Sephadex G-50 fine equilibrated with PEM containing 0.1 m M GTP and 2.0 m M DTE. The resulting tubulin preparation will assemble into microtubules when warmed to 37 ° in the presence of 2 M glycerol and 1.0 m M GTP. Acknowledgments I wish to thank Dr. Joseph Culotti and Dr. Robert Holmgren for support, encouragement, and laboratory space, Fraydoon Rastinejad for assistance, and Dr. Stephanie Jones for helpful comments on this chapter.
[25] M i c r o t u b u l e s , T u b u l i n , a n d M i c r o t u b u l e - A s s o c i a t e d Proteins of Trypanosomes
By DERRICK ROBINSON, PAULINE BEATTIE, TREVOR SHERWIN, and I~ITH
GULL
Organisms African trypanosomes possess a fascinating life cycle during which the cells undergo a variety of modulations of cell shape and motility. The precise form of the cell, its division, and motility are a reflection of the highly organized internal microtubule cytoskeleton. Although many trypanosome species are studied for their parasitological interest, aspects of their microtubule biology and motility are best advanced in Trypanosoma brucei and Crithidia fasciculata, both of which are amenable to easy cultivation in vitro. Crithidiafasciculata, a common parasite of mosquitoes, has long been used as a model organism in that it is nonpathogenic and is easily grown in a variety of simple media. ~ It grows readily to cell densities of around 1D. Evans, in "Methods of Cultivating Parasites in vitro" (A.E.R. Taylor and J. R. Baker, eds.), p. 62. Academic, New York, 1978.
METHODS IN ENZYMOLOGY, VOL 196
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4 X 107/ml and is maintained in these liquid media by simple subculturing. It is a hardy, unfastidious experimental organism. Much progress has been made recently on developing the important pathogenic African trypanosomes as amenable laboratory organisms. This trend represents fusion of interests whereby basic studies of the molecular and cellular biology of the organisms are providing direct insights to their remarkable and devastating host-parasite relationships. The African trypanosome life cycle is complex, alternating between a period of development in the bloodstream of a mammalian host, followed by a spell of maturation in the tsetse fly vector. This cycle, exemplified by T. brucei has been extensively reviewed in the literature. 2-4 Strains of T. brucei are available which are noninfective to humans and may be easily grown in liquid cultures in the laboratory using simple microbiological techniques. These culture forms of T. brucei exhibit the characteristics of the procyclic form found naturally in the insect vector midgut. Many of these procyclic strains such as 4275 and STIB 3666 have been in continuous axenic culture for long periods. Strains such as these grow well in a medium, SDM 79, 7 which resembles a supplemented mammalian tissue culture medium. Trypanosomes are grown in tissue culture flasks (12-ml medium volume in a 25-cm2 flask or 50 ml in a 75-cm2 flask) in a normal laboratory incubator at 27 °. Cell growth is quantified by direct cell counts using a counting chamber and general observations of the intensely motile cells are made using an inverted microscope in a manner analogous to mammalian cells. Cells are passaged every 2 - 3 days by inoculating at low density (approximately 50 gl of a late log-phase culture) and they grow to cell densities of 3 × 107/ml in a few days. The mean generation time is around 8.5 hr and details of the cell division cycle have recently been described in Sherwin and Gull s and Woodward and Gull. 9 Other media are available for specific experimental procedures: these include ME83, suitable for radiolabeling experiments, and HHP84, which is a fully defined medium which supports cell viability and motility but not proliferation. ~° If there is a specific requirement to use the bloodstream forms of T. brucei these can be maintained in vivo by subpassage through laboratory 2 C. A. Hoare, in "The Trypanosomes of Mammals," p. 749. BlackweU, Oxford, 1972. 3 K. Vickerman, Br. Med. Bull. 41, 105 (1985). 4 K. Vickerman, L. Tetley, K. A. K. Hendry, and C. M. R. Turner, Biol Cell, 64, 109 (1988). 5 R. Sasse and K. Gull, J. CelISci. 90, 577 (1988). 6 T. Seebeck, A. Schneider, V. Kueng, K. Schlaeppi, and A. Hemphill, Protoplasma 145, 188 (1988). 7 R. Brun and M. Schonenberger, Acta Trop. 36, 289 (1979). s T. Sherwin and K. Gull, Philos. Trans. R. Soc. (London), Set. B 323, 573 (1989). 9 R. Woodward and K. Gull, J. Cell Sci. 95, 49 (1990). io T. Seebeck and V. Kurath, Acta Trop. 42, 127 (1985).
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rodents. Parasitemias of around 109/ml can be reached and the trypanosomes can be purified from the blood using a DEAE column technique. H Trypanosome Cytoskeleton Microtubules compose the most prominent part of the trypanosome cytoskeleton; however, unlike those of the mammalian cell, these microtubules are precisely cross-linked to form two defined structures. The main cell body of the trypanosome is encircled by a highly cross-linked corset of subpellicular microtubules (Fig. l a). These microtubules determine the shape of the cell and are present throughout the complete cell cycle, during which the cell inserts new microtubules and partitions the resulting cytoskeleton to the two daughter cells. The second microtubule array is that of the flagellum basal-body-axoneme complex. The T. brucei cell possesses a single flagellum that attaches along the length of the cell body and follows a left-handed helical path originating from a flagellum pocket at the posterior end of the cell (Fig. la). The flagellum contains a classical 9 + 2 axoneme together with a paraflagellar rod, linked by an electron-dense connection to the B subfiber of microtubule doublet 7 (Fig. lb). Another connection is then made from the paraflagellar rod to the internal face of the flagellar membrane. Directly opposite this connection, a specialized zone of filaments underlies the plasma membrane of the cell body interrupting the corset of subpellicular microtubules. Four particular microtubules, which are always intimately associated with a portion of smooth endoplasmic reticulum, are invariably positioned on the immediate lefthand side of the flagellar attachment zone when viewed from the posterior of the cell (Fig. lb). The subpellicular microtubules possess a regular spacing of 18-22 nm and have a highly ordered array of side arms. s This precisely ordered array of microtubules therefore provides a very useful model in which microtubule-associated proteins are envisaged to perform roles in microtubule-microtubule and microtubule-membrane linkage, together with motility functions associated with the anfractuous movements of the trypanosome cell body.
Isolation and Characterization of Cytoskeleton The highly cross-linked nature of the trypanosome cytoskeleton allows it to be isolated intact via a simple detergent extraction procedure. The resulting microtubule cytoskeleton can be visualized in both the light and electron microscope and retains the overall shape and form of the original cell. H S. M. Lanham and D. G. Godfrey, Exp. Parasitol. 28, 521 (1970).
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FIG. 1. A transmission electron micrograph of a detergent-extracted, negatively stained cytoskeleton of T. brucei is shown in (a) (bar: 1.1/lm). The subpellicular array of microtubules is deafly visible and the flagellum appears as an electron-dense rodlike structure running along the whole length of the cell. A transverse section of the flagellum and the
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These cytoskeleton preparations provide a very useful starting point for protein purification since they facilitate an easy enrichment for microtubular structures with a consequential elimination of cytoplasmic material. However, there are important caveats to this procedure in that is now known that certain types of proteins may exhibit artifactual binding to these preparations and so masquerade as microtubule-associated proteins (MAPs). 12 Protocolfor Cytoskeleton Preparation. The preparation of trypanosome cytoskeletons is achieved by the extraction of whole cells with a nonionic detergent in a suitable buffer. The detergent solubilizes the plasma membrane and the internal membranous organelles of the trypanosome, leaving the microtubule cytoskeleton which is stabilized by the presence of EGTA and magnesium ions in the buffer. Two nonionic detergents are commonly used for this extraction: Triton X-100 and Nonidet P-40 (NP-40). Procedure. Trypanosomes are harvested by centrifugation (l,000 g at room temperature for 5-10 min), washed once in phosphate-buffered saline, and collected by centrifugation. The cell pellet is gently resuspended in either of the two detergent-buffer solutions detailed below. After incubation for a short period the cytoskeletons are collected by centrifugation and may be washed once more in the detergent-buffer solution. The cytoskeletons can be protected by including a mix of protease inhibitors in the buffer solutions [leupeptin, 50/tg/ml; pepstatin, 5/tg/ml; chymostatin, 5/tg/ml; and phenylmethylsulfonyl fluoride (PMSF), 5/tg/ml]. Triton X-IO0 method." Cell pellets may be resuspended in Triton X-100 (0.2-0.5%) in MME buffer [10 m M (3-[N-Morpholino]propanesulphonic acid) (MOPS), pH 6.9, 2 m M EGTA, l m M magnesium sulfate] and incubated on ice for 1 - 10 min. ~3 Nonidet P-40 method."Cell pellets may be resuspended in Nonidet P-40 (1%) in PEME buffer (0.1 M PIPES, pH 6.9, 2 mMEGTA, 1 mMmagnesium sulfate, 0.1 m M EDTA) and incubated at room temperature for 5 min. s,~4,~5An electron micrograph of a T. brucei cytoskeleton extracted by this method and then negatively stained is shown in Fig. 1a. t2 M. Parsons and J. M. Smith, Nucleic Acids Res. 17, 15 (1989). 13A. Schneider, H. U. Lutz, R. Marugg, P. Gehr, and T. Seebeck, J. CellSci. 90, 307 (1988). adjacent region of the subpellicular array of microtubules is depicted in (b) (bar: 70 nm). The 9 + 2 axoneme is accompanied in the flagellum by the paraflagellar rod (PFR). The connections between the axoneme and PFR and between the PFR and the plasma membrane are visible (arrows). The four subpellicular microtubules associated with a portion of the smooth endoplasmic reticulum (ER) (arrow heads) and the filamentous flagellum attachment zone (square bracket) are also visible in the cell body.
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Uses of Cytoskeletons Cytoskeletons obtained by the above methods have proved extremely useful in biochemical analysis of the tubulin isoform constitution of T. bruce?4,~6 and also of other proteins composing elements such as the paraflagellar rod. ~3,~7a8These detergent-extracted cytoskeletons have also proved extremely useful as complex immunogens for the production of monoclonal antibodies to known and cryptic elements of the eytoskeleton.19 Electron Microscopy Visualization of the Cytoskeleton The complete cytoskeleton of trypanosomes can be visualized by electron microscopy. This technique using whole-mount cytoskeletons has proved invaluable for ultrastructural characterization of cytoskeletal elements and for immunogold probing of these elements with specific antibodies.
Ultrastructural Examination of the Cytoskeleton This technique uses a fast, simple method of detergent extraction oftbe trypanosome cytoskeleton followed by fixation and negative staining) A typical cytoskeleton obtained from this procedure is depicted in Fig. 1a.
Materials Phosphate-buffered saline (PBS) (137 mM NaCI, 3 mM KC1, 7 mM Na2HPO4" 12H20, 1 mM KH2PO4, pH 7.4) Nonidet P-40 PEME buffer (detailed earlier) Glutaraldehyde (EM grade) Gold thioglucose Procedure 1. Trypanosome cells are harvested from a mid-log culture and resuspended to half the original volume in PBS. L4T. Sherwin, A. Schneider, R. Sasse, T. Seebeck, and K. Gull, J. CellBiol. 104, 439 (1987). 15T. Sherwin and K. Gull, Cell (Cambridge, Mass.) 57, 211 (1989). ~6A. Schneider, T. Sherwin, R. Sasse, D. G. Russell, and K. Gull, J. Cell Biol. 104, 431 0987). ~7D. G. Russell, R. J. Newsam, G. C. N. Palmer, and K. Gull, Eur. J. Cell Biol. 30, 137 (1983). 18j. M. Gallo and J. Schrevel, Eur. J. Cell Biol. 36, 163 (1985). ~9A. Woods, T. Sherwin, R. Sasse, T. H. MacRae, A. J. Baines, and K. Gull, J. Cell Sci. 93, 491 (1989).
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2. A charged carbon, Formvar-coated EM grid is floated on top of a drop of the dense cell suspension for 1 - 2 min. 3. Transfer the grid to a drop of I% Nonidet P-40 in PEME for 1-5 min. 4. Transfer the grid to a drop of 2.5% glutaraldehyde in PEME for 30 see.
5. Negatively stain the grid with 20/zl of 0.7% gold thioglucose in distilled water.
ImmunogoM Probing of Cytoskeletons The subcellular localization of cytoskeletal antigens can be visualized by the technique of immunogold labeling of whole-mount cytoskeletons of
T. brucei.15 Materials Phosphate-buffered saline (PBS) Nonidet P-40 PEME buffer Paraformaldehyde Glycine Bovine serum albumin (BSA) Gold-conjugated second antibody Glutaraldehyde Ammonium molybdate.
Procedure 1. Trypanosome cells are harvested and resuspended in PBS to half the original volume. 2. A charged carbon, Formvar-coated EM grid is floated on top of a drop of the cell suspension for 3 rain. 3. Transfer the grid to a drop of 1% Nonidet P-40 in PEME for 1-5 min. 4. Fix the grid on a drop of 3.7% paraformaldehyde in PEME for 15 min. 5. Wash grid in PEME. 6. Neutralize free aldehyde groups on a drop of 20 m M glycine in PBS for 5 min. 7. Block nonspecific binding by transfer to 1% BSA in PBS for 5 min. 8. Incubate grid in first antibody diluted in 1% BSA in PBS for 45 min at room temperature in a moist atmosphere. 9. Wash grid three times for 5 min each time in 1% BSA in PBS.
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10. Incubate in second antibody (gold conjugate) diluted in 1% BSA in PBS as for the first incubation. 11. Wash once for 5 min in 1% BSA in PBS. 12. Wash three times for 5 min each time in 0.1% BSA in PBS. 13. Wash three times for 5 min each time in PEME. 14. Fix in 2.5% glutaraldehyde in PEME. 15. Negatively stain the grid using 2% ammonium molybdate, pH 7.0. Alternatively to the negative staining (step 15) grids may be positively stained with 2% ammonium molybdate, washed in water, dehydrated through an acetone series, and critically point dried.
Fractionation of Trypanosome Cytoskeletons The two microtubular structures of the cell, the flagellum and subpellicular corset, can be dissected from each other, allowing the organelles to be studied in isolation. Depolymerization of the pellicular microtubules can be achieved using a high-salt concentration or by the action of calcium ions, thus leaving the flagellum intact. ~6,2°
Selective Depolymerization of Subpellicular Microtubules and Isolation of Flagella Materials Triton X- 100 PMN buffer: 10 m M sodium phosphate, pH 7.2, 150 m M sodium chloride, 1 m M magnesium chloride Calcium chloride (1 mM) Sodium chloride (1 M) Procedure. Trypanosoma brucei cells are harvested and washed in phosphate-buffered saline (PBS). The resulting pellet of cells is resuspended in PMN buffer containing 0.5% Triton X-100. The cytoskeletons obtained from this treatment are washed twice in the same buffer. Following this, one of two treatments may be performed. 1. Cytoskeletons are resuspended in PMN buffer containing 0.5% Triton X-100 and 1 m M calcium chloride and incubated on ice for 45 min. Centrifugation of the suspension at 100,000 g at 4 ° for 1 hr produces a supernatant that is highly enriched in subpellicular tubulin.~6 2. The cytoskeletons are resuspended in PMN buffer containing 1 M 20 M. T. Dolan and C. G. Reid, J. CellSci. 80, 123 (1986).
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sodium chloride. After incubation on ice for 10 rain a high-speed spin once again leaves the supernatant enriched in pellicular tubulin.16 Selective depolymerization of the subpellicular microtubules using either of the above methods leaves the flagellum intact. Centrifugation of the incubation mixture at 16,000 g for 10 min pellets the flagella and the remaining supernatant can be discarded, or spun at 100,000 g in order to study the solubilized pellicular fraction as above. The pellet of flagella can be washed in the depolymerization buffer to ensure complete removal of the corset microtubules.16 The cytoskeletons and flagella can be visualized under phase-contrast microscopy and the preparations should be constantly monitored by this technique to ensure the extent of extraction and quality of the fractions.
T r y p a n o s o m e Tubulin Genes and Isoforms Trypanosomes possess multiple genes for both or- and fl-tubulin, although the available evidence suggests that all of the a-tubulin genes are identical, as are all of the fl-tubulin genes. 21 In T. brucei the tubulin genes are arranged in a tightly packed cluster of alternating ot and fl genes. In T. brucei there are thought to be approximately 10-15 copies of each gene type arranged in this manner, 22,23 whereas in C. fasciculata tubulin genes have a dispersed arrangement. 24 During expression in T. brucei the tubulin gene duster is transcribed as a single polycistronic unit. 25 This single transcript is then trans-spliced to produce mature mRNA. 26,27During this process only a single species each of a-tubulin mRNA and fl-tubulin mRNA is produced. 2s,29In vitro translation of hybrid-selected ot-tubulin mRNA results in a single translation product when observed by two-dimensional (2D) gel electrophoresis) 6 21 B. E. Kimmel, S. Samson, J. Wu, R. Hirschberg, and L. R. Yarborough, Gene 35, 237 (1985). 22 T. Seebeck, P. Whittaker, M. A. Imboden, N. Hardman, and R. Braun, Proc. NatL Acad. Sci. U.S.A. 80, 4634 (1983). 23 L. S. Thomashow, M. Milhausen, W. J. Rutter, and N, Agabian, Cell (Cambridge, Mass.) 32, 35 (1983). 24 I. Tittawella and S. Normark, FEMSMicrobiol. Lett. 43, 317 (1987). 25 M. A. Imboden, P. W. Laird, M. Affolter, and T. Seebeck, Nucleic Acids Res. 15, 7357 (1987). 26 p. Borst, Annu. Rev. Biochem. 55, 701 (1986). 27 R. Braun, Bioessays 5, 223 (1986). 2s M. A. Imboden, B. Blum, T. de Lange, R. Braun, and T. Seebeck, J. Mol. Biol. 188, 293 (1986). 29 S. Sather and N. Agabian, Proc. Natl. Acad. Sci. U.S.A. 82, 5695 (1985).
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However, 2D gel analysis of the tubulins isolated initially from C. fasciculata, and confirmed for T. bruceL reveals two distinct ce-tubulin isoforms. Subsequent biochemical and immunological analysis has shown that the primary ot-tubulin translation product can be modified via two separate, reversible posttranslational processes: tubulin acetylation and detyrosination.5,14-16
Isolation and Purification of Tubulin The purification of trypanosome tubulin has allowed the identification of the tubulin isoforms and their cellular distribution, and also provides an opportunity to develop assays to reveal the role of MAPs in the construction of the cytoskeleton. Although mammalian tubulin is more easily purifiable in larger amounts, there are some arguments to suggest that the use of a homologous tubulin may have advantages in binding, assembly, cross-linking, or motility assays. Russell et al. 3° developed procedures for the isolation, purification, and in vitro polymerization of tubulin from the flagellum, the subpellicular array, and the cytoplasmic pool of C. fasciculata. Flagella were isolated by mechanical agitation and purified on a sucrose gradient. After demembranation the axonemal microtubules were depolymerized by extensive dialysis. The solubilized tubulin was concentrated by glycerol dialysis and assembled. Cell bodies from the above disruption were detergent extracted and sonicated further to depolymerize the subpellicular microtubules. The solution was then cleared by centrifugation and concentrated by dialysis against glycerol. After clarification by centrifugation the dialyzate was taken through two cycles of assembly and disassembly. Assembly-competent tubulin was obtained from the cytoplasmic pool by using a column purification protocol. Cells were harvested, washed in buffer, and then lysed by gentle sonication. The cell bodies were removed by centrifugation and the supernatant run on a DEAE-Sephadex ion-exchange colomn. Tubulin was eluted using a salt cut of 0.55 M KC1, concentrated in an Amicon (Danvers, MA) concentrator, cleared by centrifugation, dialyzed at 4 °, and then polymerized at 37 ° for 30 min. Although this study developed the protocols for tubulin purification and polymerization the tubulin from all three fractions did not form complete microtubules. Rather, the tubulin polymerized into sheets and 3oD. G. Russell, D. Miller, and K. Gull, Mol. Cell Biol. 4, 779 (1984).
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ribbons. These protocols have since been used and adapted by Bramblett et a/. 3~,32to isolate putative MAPs. Stieger et al. 33 were able to partially purify the characterize tubulin from T. brucei by use of taxol-mediated assembly. Essentially, cells were lysed by sonication and centrifuged. Tubulin in the 100,000 g supernatant was assembled into microtubules by incubation at 30 ° in the presence of taxol and GTP. Recently, a general method has been developed which allows the complete purification of tubulin from T. brucei and its assembly in vitro into structurally normal microtubules.34 The technique involves the purification of tubulin by DEAE-Sephadex chromatography, Amicon concentration, glycerol dialysis, and then polymerization in vitro. The protocol is summarized below. 1. Cells grown to a density of 2 - 4 × 107/ml are harvested from 2 liters of medium by centrifugation at 1400 g for 5 min at 4 °. 2. Wash once in PEME supplemented with 4 M glycerol, 0.1 m M GTP, and 50/zg of leupeptin/ml, pH 6.9. 3. The washed cells are resuspended in 6 ml of the supplemented PEME, passed through a French press, and sonicated for four 30-sec periods, interspersed with 2-min incubations in an ice/water bath. 4. The sonicated cells are incubated on ice for 30 min, then centrifuged at 40,000 g for 30 min at 4 °. The supernatant is recentrifuged under the same conditions for 20 min. The resulting supernatant can be used immediately or stored at - 8 0 °. 5. The cell-free supernatant is applied at a flow rate of 25 ml/hr to a 28-mi DEAE-Sephadex column packed in a 30-ml disposable syringe, equilibrated with PEME containing 0.2 M KC1, 0.1 m M GTP, plus 12.5 /tg/ml of leupeptin. 6. The column is washed with the equilibration buffer and adherent protein is eluted with PEME containing 0.6 M KC1, 0.1 m M GTP, and 12.5/~g/ml leupeptin. Peak fractions are pooled and can be used immediately or stored at - 8 0 °. 7. The 0.6 M KC1 fraction from the column is concentrated to 2 - 3 ml in an Amicon ultrafiltration assembly. After filtration the preparation is further concentrated by dialysis overnight against 125 ml of PEME containing 8 M glycerol, 0.1 m M GTP, and 25/tg/ml leupeptin at 4* 3~G. T. Bramblett, S. C. Chang, and M. Flavin, Proc. Natl. Acad. Sci. U.S.A. 84, 3259 (1987). 32G. T. Bramblett, R. Kambadur, and M. Flavin, CellMot. Cytoskeleton 13, 145 (1989). 33j. Stieger, T. Wyler, and T. Seebeck, J. Biol. Chem. 259, 4596 (1984). T. H. MacRae and K. Gull, Biochem. J. (Tokyo) 265, 87 (1990).
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8. The dialyzed tubulin is centrifuged (40,000 g) twice (for 30 min and then 20 min) at 4 °, polymerized at 37 ° for 30 min in the presence of 10 m M Mg2+ and 1.8 m M GTP. The assembled tubulin is pelleted by centrifugation (40,000 g at 20 °) for 30 min. Since the microtubules pack poorly only 70% of the supernatant is removed and the loose pellet is resuspended in 50/A of PEME containing 1.8 m M GTP and incubated for 15 min at 37 °. 9. The preparation is centrifuged again at 40,000 g at 20°C. The supernatant is removed and the surface of the pellet washed with 100 #1 of PEME containing 0.1 m M G T P at 37 °. The pellet is resuspended in 200/zl of PEME containing 0.1 m M GTP and depolymerized by incubation on ice for 30 min, with gentle vortex mixing. 10. The suspension is then centrifuged at 40,000g for 30 min at 4°. The resulting supernatant containing the purified tubulin (final yield 0.9 mg) is stored at - 70 °. Trypanosome tubulin purified in the above manner assembles readily at a concentration of 2 mg/ml in the presence of 1.8 m M GTP at 37 °.
Microtubule-Associated Proteins in Trypanosomes The notable feature of the trypanosome cytoskeleton is the regularly spaced helical array of subpellicular microtubules. Ultrastructural studies have shown that the periodicity of these microtubules and their association with the plasma membrane is retained by a series of intermicrotubule cross-bridges and microtubule-membrane linkages. This has led many workers to search for the microtubule-associated proteins (MAPs) responsible for controlling such phenomena in trypanosomes. To date, a handful of candidate MAPs have been described. These putative MAPs are listed below together with a short description of their properties. A number of approaches have been taken to facilitate identification of subpellicular MAPs. Clearly, some of these have proved more effective than others and we will discuss the merits and disadvantages of the approaches adopted. The history of the identification of trypanosome MAPs illustrates the difficulties, yet necessity, of providing a precise definition of the term MAP. It is clear that some proteins identified as putative MAPs by their ability to associate with microtubules in vitro (in the detergent-extracted cytoskeleton or with reassembled microtubules) have turned out to be glycosomal enzymes. ~2,32These reports emphasize the requirement to work toward a more extensive definition of a putative MAP in terms of its behavior in vitro, its position within the intact cell, and preferably its molecular identity.
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MAP Fraction from Crithidia fasciculata A series of cytoplasmic MAPs were identified in C fasciculala. 31 These MAPs were obtained by purifying cytoplasmic microtubule protein as described earlier,3° and polymerizing stable microtubules in the presence of taxol and dimethyl sulfoxide. The MAPs were solubilized by extraction of the pellet with 0.6 M sodium chloride buffer while the microtubules were stabilized with taxol. After concentration by centricon filter and dialysis, several proteins were present in the enhanced fraction. The most notable proteins in this fraction possessed molecular weights of 130K, 90K, 65K, 49K, 36K, and 33K. Incubation of this fraction with tubulin induced microtubule polymerization, although it was 10 times less effective than the MAP fraction isolated from mammalian brain. Two of the trypanosome proteins, MAP33 and MAP40, were concentrated in the polymer fraction. These authors 31,32also isolated a set of three proteins (corset proteins, COPs) which were associated with the microtubule corset of C. fasciculata. Essentially pellicular microtubular protein was isolated from this organism as described earlier, 3° induced to assemble by dialysis against glycerol, and the microtubuIes discarded. The remaining fraction contained the COPs. The proteins were then fractionated by salt gradient elution from a cationexchange Mono S column. The three proteins isolated by this method were termed COP41, COP61, and COP33 (the numbers referring to their apparent molecular weights). COP41. This protein, when incubated with polymerized brain tubulin, formed cross-links which were periodic along the lengths of the microtubules. Also, the native protein appeared to be tetrameric with a molecular weight of approximately 160K. However, immunogold localization of this protein in C. fasciculata revealed that an antibody specific for this protein bound only to glycosomes. Upon further investigation COP41 proved to be glyceraldehyde 3-P-dehydrogenase (GAPDH). Thus, in cells this enzyme was confined in glycosomes and only bound to corset microtubules after release by homogenization. COP61. This protein, which forms a dimer of Mr 120K, bound avidly .to preassembled microtubules but did not bundle or cross-link them. Immunogold localization with a polyclona! antibody to COP61 suggested that the antibody showed some affinity for cytoskeletons when incubated with them as whole,mounts, but no clear affinity for microtubules in intact cells. COP33. This protein readily formed cross-links between preassembled
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microtubules and is thought to form a tetramer of Mr 135K. The crosslinks between the microtubules had a periodicity of 8.5 nm. However, a polyclonal antibody raised to COP33 showed no specific binding to cytoskeletons or cell sections. Thus, again the identification of this protein as a MAP requires confirmation. MAPs from Trypanosoma brucei The search for trypanosomal MAPs has also produced some candidates from T. brucei. p60. A protein of M, 60K was isolated from T. brucei by Seebeck et al. 35 The protein was purified by a succession of gel-filtration and ion-exchange chromatography or by velocity sedimentation in a glycerol gradient from a crude cytoskeletal extract. 1360was found to (1) copolymerize with tubulin, (2) bind to preassembled microtubules, (3) bind to synthetic liposomes, and (4) cross-link microtubules and membrane vesicles. All this seemed to suggest that p60 was a trypanosomal MAP. However, it now appears that p60 is in fact a glycosomal protein, phosphoenolpyruvate carboxykinase. 12.36 p41. This protein was isolated from T. brucei cytoskeletons by extraction with 0.1% Triton X-100 and 1 m M EGTA. 37 The protein, once isolated, was found to possess covalently bound fatty acid. 1341 was found to remain tightly bound to the cytoskeleton if calcium ions were present, but it could also be selectively released if cytoskeletons were incubated in excess EGTA. Purified 1341 was found to bind to isolated cytoskeletons and to preassembled microtubules of trypanosome tubulin. p320. p320 is a heat-stable protein which was isolated from the pellicular tubulin fraction of T. brucei by heat treatment of the 0.75 M sodium chloride-solubilized fraction of the cytoskeleton.36 It was localized to the pellicular microtubules of T. brucei by immunogold probing with a specific polyclonal antibody, p320 also polymerized with tubulin when trypanosome cell lysates were induced to assemble by taxol. A cloned segment of the p320 gene indicated that the predicted protein sequence consisted of 50 nearly identical tandem repeats of 38 amino acids. To date this remains the most promising and best described candidate for a trypanosomal MAP. 52K Protein. Solubilization of the subpellicular microtubules of T. brucei by a high-strength salt solution, and further fractionation by Mono S
3s T. Seebeck, V. Kueng, T. Wyler, and M. Muller, Proc. Natl, Acad. Sci. U.S.A. 85, 1101 (1988). 36A. Schneider, A. Hemphill, T. Wyler, and A. Seebeck, Science 241, 459 (1988). 37A. Schneider, W. Eichenberger, and T. Seebeck, J. Biol. Chem. 263, 6472 (1988).
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cation-exchange column chromatography, led to an Mr 52K protein being eluted. 3a The 52K protein, when bound to nitrocellulose, gave an indication of tubulin binding. Also, when this protein was incubated with brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular cross-links between the microtubules. However, there is no biochemical or immunological characterization of this protein, nor information as to its location in the intact trypanosome cell. Again, therefore, its identification as a defined MAP must await further validation. We now possess a rather extensive knowledge of the structural components and ultrastructural organization of the trypanosome cytoskeleton. We also have a good understanding of the molecular biology and cell biology of the major structural protein, tubulin. The precise shape and form of this cell makes it an extremely suitable system in which to study the assembly of cytoskeletal structures possessing very high spatial order. The regulatory, interacting proteins responsible for the assembly maintenance, and functional-properties of the cytoskeleton are likely to be of great general interest. We have only just started to gain an insight into the identity of these proteins and it is clear that our best understanding of their roles will come from multifaceted studies which include molecular, cellular, and biochemical approaches. Acknowledgments Work in this laboratoryreceivedfinancialsupport from the UNDP/WorldBank/WHO special program for Researchand Training in Tropical Diseases. D.R.R. was funded by a SERC studentshipand P.B. was fundedby a WellcomePrizestudentship.
3s N. Balaban, H. K. Waithaka, A. R. Njogu, and R. Goldman. Cell Mot. Cytoskeleton 14, 393 (1989).
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preequilibrated with C buffer. The tubulin will bind to the DEAE. Wash the column with 200 ml of C buffer containing 0.35 M NaCI and then elute the tubulin from the column with C buffer containing 0.65 M NaCI. Add 0.38 g/ml ammonium sulfate to the tubulin containing fractions and mix gently. Leave on ice for 20 rain and centrifuge at 27,000 g for 20 min at 0 ° to precipitate the tubulin. Resuspend the pellet to a concentration of 4.0 mg/ml in PEM buffer containing 0.1 m M GTP and 2.0 m M DTE and desalt the tubulin on a spin column containing Sephadex G-50 fine equilibrated with PEM containing 0.1 m M GTP and 2.0 m M DTE. The resulting tubulin preparation will assemble into microtubules when warmed to 37 ° in the presence of 2 M glycerol and 1.0 m M GTP. Acknowledgments I wish to thank Dr. Joseph Culotti and Dr. Robert Holmgren for support, encouragement, and laboratory space, Fraydoon Rastinejad for assistance, and Dr. Stephanie Jones for helpful comments on this chapter.
[25] M i c r o t u b u l e s , T u b u l i n , a n d M i c r o t u b u l e - A s s o c i a t e d Proteins of Trypanosomes
By DERRICK ROBINSON, PAULINE BEATTIE, TREVOR SHERWIN, and I~ITH
GULL
Organisms African trypanosomes possess a fascinating life cycle during which the cells undergo a variety of modulations of cell shape and motility. The precise form of the cell, its division, and motility are a reflection of the highly organized internal microtubule cytoskeleton. Although many trypanosome species are studied for their parasitological interest, aspects of their microtubule biology and motility are best advanced in Trypanosoma brucei and Crithidia fasciculata, both of which are amenable to easy cultivation in vitro. Crithidiafasciculata, a common parasite of mosquitoes, has long been used as a model organism in that it is nonpathogenic and is easily grown in a variety of simple media. ~ It grows readily to cell densities of around 1D. Evans, in "Methods of Cultivating Parasites in vitro" (A.E.R. Taylor and J. R. Baker, eds.), p. 62. Academic, New York, 1978.
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4 X 107/ml and is maintained in these liquid media by simple subculturing. It is a hardy, unfastidious experimental organism. Much progress has been made recently on developing the important pathogenic African trypanosomes as amenable laboratory organisms. This trend represents fusion of interests whereby basic studies of the molecular and cellular biology of the organisms are providing direct insights to their remarkable and devastating host-parasite relationships. The African trypanosome life cycle is complex, alternating between a period of development in the bloodstream of a mammalian host, followed by a spell of maturation in the tsetse fly vector. This cycle, exemplified by T. brucei has been extensively reviewed in the literature. 2-4 Strains of T. brucei are available which are noninfective to humans and may be easily grown in liquid cultures in the laboratory using simple microbiological techniques. These culture forms of T. brucei exhibit the characteristics of the procyclic form found naturally in the insect vector midgut. Many of these procyclic strains such as 4275 and STIB 3666 have been in continuous axenic culture for long periods. Strains such as these grow well in a medium, SDM 79, 7 which resembles a supplemented mammalian tissue culture medium. Trypanosomes are grown in tissue culture flasks (12-ml medium volume in a 25-cm2 flask or 50 ml in a 75-cm2 flask) in a normal laboratory incubator at 27 °. Cell growth is quantified by direct cell counts using a counting chamber and general observations of the intensely motile cells are made using an inverted microscope in a manner analogous to mammalian cells. Cells are passaged every 2 - 3 days by inoculating at low density (approximately 50 gl of a late log-phase culture) and they grow to cell densities of 3 × 107/ml in a few days. The mean generation time is around 8.5 hr and details of the cell division cycle have recently been described in Sherwin and Gull s and Woodward and Gull. 9 Other media are available for specific experimental procedures: these include ME83, suitable for radiolabeling experiments, and HHP84, which is a fully defined medium which supports cell viability and motility but not proliferation. ~° If there is a specific requirement to use the bloodstream forms of T. brucei these can be maintained in vivo by subpassage through laboratory 2 C. A. Hoare, in "The Trypanosomes of Mammals," p. 749. BlackweU, Oxford, 1972. 3 K. Vickerman, Br. Med. Bull. 41, 105 (1985). 4 K. Vickerman, L. Tetley, K. A. K. Hendry, and C. M. R. Turner, Biol Cell, 64, 109 (1988). 5 R. Sasse and K. Gull, J. CelISci. 90, 577 (1988). 6 T. Seebeck, A. Schneider, V. Kueng, K. Schlaeppi, and A. Hemphill, Protoplasma 145, 188 (1988). 7 R. Brun and M. Schonenberger, Acta Trop. 36, 289 (1979). s T. Sherwin and K. Gull, Philos. Trans. R. Soc. (London), Set. B 323, 573 (1989). 9 R. Woodward and K. Gull, J. Cell Sci. 95, 49 (1990). io T. Seebeck and V. Kurath, Acta Trop. 42, 127 (1985).
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rodents. Parasitemias of around 109/ml can be reached and the trypanosomes can be purified from the blood using a DEAE column technique. H Trypanosome Cytoskeleton Microtubules compose the most prominent part of the trypanosome cytoskeleton; however, unlike those of the mammalian cell, these microtubules are precisely cross-linked to form two defined structures. The main cell body of the trypanosome is encircled by a highly cross-linked corset of subpellicular microtubules (Fig. l a). These microtubules determine the shape of the cell and are present throughout the complete cell cycle, during which the cell inserts new microtubules and partitions the resulting cytoskeleton to the two daughter cells. The second microtubule array is that of the flagellum basal-body-axoneme complex. The T. brucei cell possesses a single flagellum that attaches along the length of the cell body and follows a left-handed helical path originating from a flagellum pocket at the posterior end of the cell (Fig. la). The flagellum contains a classical 9 + 2 axoneme together with a paraflagellar rod, linked by an electron-dense connection to the B subfiber of microtubule doublet 7 (Fig. lb). Another connection is then made from the paraflagellar rod to the internal face of the flagellar membrane. Directly opposite this connection, a specialized zone of filaments underlies the plasma membrane of the cell body interrupting the corset of subpellicular microtubules. Four particular microtubules, which are always intimately associated with a portion of smooth endoplasmic reticulum, are invariably positioned on the immediate lefthand side of the flagellar attachment zone when viewed from the posterior of the cell (Fig. lb). The subpellicular microtubules possess a regular spacing of 18-22 nm and have a highly ordered array of side arms. s This precisely ordered array of microtubules therefore provides a very useful model in which microtubule-associated proteins are envisaged to perform roles in microtubule-microtubule and microtubule-membrane linkage, together with motility functions associated with the anfractuous movements of the trypanosome cell body.
Isolation and Characterization of Cytoskeleton The highly cross-linked nature of the trypanosome cytoskeleton allows it to be isolated intact via a simple detergent extraction procedure. The resulting microtubule cytoskeleton can be visualized in both the light and electron microscope and retains the overall shape and form of the original cell. H S. M. Lanham and D. G. Godfrey, Exp. Parasitol. 28, 521 (1970).
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FIG. 1. A transmission electron micrograph of a detergent-extracted, negatively stained cytoskeleton of T. brucei is shown in (a) (bar: 1.1/lm). The subpellicular array of microtubules is deafly visible and the flagellum appears as an electron-dense rodlike structure running along the whole length of the cell. A transverse section of the flagellum and the
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These cytoskeleton preparations provide a very useful starting point for protein purification since they facilitate an easy enrichment for microtubular structures with a consequential elimination of cytoplasmic material. However, there are important caveats to this procedure in that is now known that certain types of proteins may exhibit artifactual binding to these preparations and so masquerade as microtubule-associated proteins (MAPs). 12 Protocolfor Cytoskeleton Preparation. The preparation of trypanosome cytoskeletons is achieved by the extraction of whole cells with a nonionic detergent in a suitable buffer. The detergent solubilizes the plasma membrane and the internal membranous organelles of the trypanosome, leaving the microtubule cytoskeleton which is stabilized by the presence of EGTA and magnesium ions in the buffer. Two nonionic detergents are commonly used for this extraction: Triton X-100 and Nonidet P-40 (NP-40). Procedure. Trypanosomes are harvested by centrifugation (l,000 g at room temperature for 5-10 min), washed once in phosphate-buffered saline, and collected by centrifugation. The cell pellet is gently resuspended in either of the two detergent-buffer solutions detailed below. After incubation for a short period the cytoskeletons are collected by centrifugation and may be washed once more in the detergent-buffer solution. The cytoskeletons can be protected by including a mix of protease inhibitors in the buffer solutions [leupeptin, 50/tg/ml; pepstatin, 5/tg/ml; chymostatin, 5/tg/ml; and phenylmethylsulfonyl fluoride (PMSF), 5/tg/ml]. Triton X-IO0 method." Cell pellets may be resuspended in Triton X-100 (0.2-0.5%) in MME buffer [10 m M (3-[N-Morpholino]propanesulphonic acid) (MOPS), pH 6.9, 2 m M EGTA, l m M magnesium sulfate] and incubated on ice for 1 - 10 min. ~3 Nonidet P-40 method."Cell pellets may be resuspended in Nonidet P-40 (1%) in PEME buffer (0.1 M PIPES, pH 6.9, 2 mMEGTA, 1 mMmagnesium sulfate, 0.1 m M EDTA) and incubated at room temperature for 5 min. s,~4,~5An electron micrograph of a T. brucei cytoskeleton extracted by this method and then negatively stained is shown in Fig. 1a. t2 M. Parsons and J. M. Smith, Nucleic Acids Res. 17, 15 (1989). 13A. Schneider, H. U. Lutz, R. Marugg, P. Gehr, and T. Seebeck, J. CellSci. 90, 307 (1988). adjacent region of the subpellicular array of microtubules is depicted in (b) (bar: 70 nm). The 9 + 2 axoneme is accompanied in the flagellum by the paraflagellar rod (PFR). The connections between the axoneme and PFR and between the PFR and the plasma membrane are visible (arrows). The four subpellicular microtubules associated with a portion of the smooth endoplasmic reticulum (ER) (arrow heads) and the filamentous flagellum attachment zone (square bracket) are also visible in the cell body.
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Uses of Cytoskeletons Cytoskeletons obtained by the above methods have proved extremely useful in biochemical analysis of the tubulin isoform constitution of T. bruce?4,~6 and also of other proteins composing elements such as the paraflagellar rod. ~3,~7a8These detergent-extracted cytoskeletons have also proved extremely useful as complex immunogens for the production of monoclonal antibodies to known and cryptic elements of the eytoskeleton.19 Electron Microscopy Visualization of the Cytoskeleton The complete cytoskeleton of trypanosomes can be visualized by electron microscopy. This technique using whole-mount cytoskeletons has proved invaluable for ultrastructural characterization of cytoskeletal elements and for immunogold probing of these elements with specific antibodies.
Ultrastructural Examination of the Cytoskeleton This technique uses a fast, simple method of detergent extraction oftbe trypanosome cytoskeleton followed by fixation and negative staining) A typical cytoskeleton obtained from this procedure is depicted in Fig. 1a.
Materials Phosphate-buffered saline (PBS) (137 mM NaCI, 3 mM KC1, 7 mM Na2HPO4" 12H20, 1 mM KH2PO4, pH 7.4) Nonidet P-40 PEME buffer (detailed earlier) Glutaraldehyde (EM grade) Gold thioglucose Procedure 1. Trypanosome cells are harvested from a mid-log culture and resuspended to half the original volume in PBS. L4T. Sherwin, A. Schneider, R. Sasse, T. Seebeck, and K. Gull, J. CellBiol. 104, 439 (1987). 15T. Sherwin and K. Gull, Cell (Cambridge, Mass.) 57, 211 (1989). ~6A. Schneider, T. Sherwin, R. Sasse, D. G. Russell, and K. Gull, J. Cell Biol. 104, 431 0987). ~7D. G. Russell, R. J. Newsam, G. C. N. Palmer, and K. Gull, Eur. J. Cell Biol. 30, 137 (1983). 18j. M. Gallo and J. Schrevel, Eur. J. Cell Biol. 36, 163 (1985). ~9A. Woods, T. Sherwin, R. Sasse, T. H. MacRae, A. J. Baines, and K. Gull, J. Cell Sci. 93, 491 (1989).
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2. A charged carbon, Formvar-coated EM grid is floated on top of a drop of the dense cell suspension for 1 - 2 min. 3. Transfer the grid to a drop of I% Nonidet P-40 in PEME for 1-5 min. 4. Transfer the grid to a drop of 2.5% glutaraldehyde in PEME for 30 see.
5. Negatively stain the grid with 20/zl of 0.7% gold thioglucose in distilled water.
ImmunogoM Probing of Cytoskeletons The subcellular localization of cytoskeletal antigens can be visualized by the technique of immunogold labeling of whole-mount cytoskeletons of
T. brucei.15 Materials Phosphate-buffered saline (PBS) Nonidet P-40 PEME buffer Paraformaldehyde Glycine Bovine serum albumin (BSA) Gold-conjugated second antibody Glutaraldehyde Ammonium molybdate.
Procedure 1. Trypanosome cells are harvested and resuspended in PBS to half the original volume. 2. A charged carbon, Formvar-coated EM grid is floated on top of a drop of the cell suspension for 3 rain. 3. Transfer the grid to a drop of 1% Nonidet P-40 in PEME for 1-5 min. 4. Fix the grid on a drop of 3.7% paraformaldehyde in PEME for 15 min. 5. Wash grid in PEME. 6. Neutralize free aldehyde groups on a drop of 20 m M glycine in PBS for 5 min. 7. Block nonspecific binding by transfer to 1% BSA in PBS for 5 min. 8. Incubate grid in first antibody diluted in 1% BSA in PBS for 45 min at room temperature in a moist atmosphere. 9. Wash grid three times for 5 min each time in 1% BSA in PBS.
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10. Incubate in second antibody (gold conjugate) diluted in 1% BSA in PBS as for the first incubation. 11. Wash once for 5 min in 1% BSA in PBS. 12. Wash three times for 5 min each time in 0.1% BSA in PBS. 13. Wash three times for 5 min each time in PEME. 14. Fix in 2.5% glutaraldehyde in PEME. 15. Negatively stain the grid using 2% ammonium molybdate, pH 7.0. Alternatively to the negative staining (step 15) grids may be positively stained with 2% ammonium molybdate, washed in water, dehydrated through an acetone series, and critically point dried.
Fractionation of Trypanosome Cytoskeletons The two microtubular structures of the cell, the flagellum and subpellicular corset, can be dissected from each other, allowing the organelles to be studied in isolation. Depolymerization of the pellicular microtubules can be achieved using a high-salt concentration or by the action of calcium ions, thus leaving the flagellum intact. ~6,2°
Selective Depolymerization of Subpellicular Microtubules and Isolation of Flagella Materials Triton X- 100 PMN buffer: 10 m M sodium phosphate, pH 7.2, 150 m M sodium chloride, 1 m M magnesium chloride Calcium chloride (1 mM) Sodium chloride (1 M) Procedure. Trypanosoma brucei cells are harvested and washed in phosphate-buffered saline (PBS). The resulting pellet of cells is resuspended in PMN buffer containing 0.5% Triton X-100. The cytoskeletons obtained from this treatment are washed twice in the same buffer. Following this, one of two treatments may be performed. 1. Cytoskeletons are resuspended in PMN buffer containing 0.5% Triton X-100 and 1 m M calcium chloride and incubated on ice for 45 min. Centrifugation of the suspension at 100,000 g at 4 ° for 1 hr produces a supernatant that is highly enriched in subpellicular tubulin.~6 2. The cytoskeletons are resuspended in PMN buffer containing 1 M 20 M. T. Dolan and C. G. Reid, J. CellSci. 80, 123 (1986).
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sodium chloride. After incubation on ice for 10 rain a high-speed spin once again leaves the supernatant enriched in pellicular tubulin.16 Selective depolymerization of the subpellicular microtubules using either of the above methods leaves the flagellum intact. Centrifugation of the incubation mixture at 16,000 g for 10 min pellets the flagella and the remaining supernatant can be discarded, or spun at 100,000 g in order to study the solubilized pellicular fraction as above. The pellet of flagella can be washed in the depolymerization buffer to ensure complete removal of the corset microtubules.16 The cytoskeletons and flagella can be visualized under phase-contrast microscopy and the preparations should be constantly monitored by this technique to ensure the extent of extraction and quality of the fractions.
T r y p a n o s o m e Tubulin Genes and Isoforms Trypanosomes possess multiple genes for both or- and fl-tubulin, although the available evidence suggests that all of the a-tubulin genes are identical, as are all of the fl-tubulin genes. 21 In T. brucei the tubulin genes are arranged in a tightly packed cluster of alternating ot and fl genes. In T. brucei there are thought to be approximately 10-15 copies of each gene type arranged in this manner, 22,23 whereas in C. fasciculata tubulin genes have a dispersed arrangement. 24 During expression in T. brucei the tubulin gene duster is transcribed as a single polycistronic unit. 25 This single transcript is then trans-spliced to produce mature mRNA. 26,27During this process only a single species each of a-tubulin mRNA and fl-tubulin mRNA is produced. 2s,29In vitro translation of hybrid-selected ot-tubulin mRNA results in a single translation product when observed by two-dimensional (2D) gel electrophoresis) 6 21 B. E. Kimmel, S. Samson, J. Wu, R. Hirschberg, and L. R. Yarborough, Gene 35, 237 (1985). 22 T. Seebeck, P. Whittaker, M. A. Imboden, N. Hardman, and R. Braun, Proc. NatL Acad. Sci. U.S.A. 80, 4634 (1983). 23 L. S. Thomashow, M. Milhausen, W. J. Rutter, and N, Agabian, Cell (Cambridge, Mass.) 32, 35 (1983). 24 I. Tittawella and S. Normark, FEMSMicrobiol. Lett. 43, 317 (1987). 25 M. A. Imboden, P. W. Laird, M. Affolter, and T. Seebeck, Nucleic Acids Res. 15, 7357 (1987). 26 p. Borst, Annu. Rev. Biochem. 55, 701 (1986). 27 R. Braun, Bioessays 5, 223 (1986). 2s M. A. Imboden, B. Blum, T. de Lange, R. Braun, and T. Seebeck, J. Mol. Biol. 188, 293 (1986). 29 S. Sather and N. Agabian, Proc. Natl. Acad. Sci. U.S.A. 82, 5695 (1985).
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However, 2D gel analysis of the tubulins isolated initially from C. fasciculata, and confirmed for T. bruceL reveals two distinct ce-tubulin isoforms. Subsequent biochemical and immunological analysis has shown that the primary ot-tubulin translation product can be modified via two separate, reversible posttranslational processes: tubulin acetylation and detyrosination.5,14-16
Isolation and Purification of Tubulin The purification of trypanosome tubulin has allowed the identification of the tubulin isoforms and their cellular distribution, and also provides an opportunity to develop assays to reveal the role of MAPs in the construction of the cytoskeleton. Although mammalian tubulin is more easily purifiable in larger amounts, there are some arguments to suggest that the use of a homologous tubulin may have advantages in binding, assembly, cross-linking, or motility assays. Russell et al. 3° developed procedures for the isolation, purification, and in vitro polymerization of tubulin from the flagellum, the subpellicular array, and the cytoplasmic pool of C. fasciculata. Flagella were isolated by mechanical agitation and purified on a sucrose gradient. After demembranation the axonemal microtubules were depolymerized by extensive dialysis. The solubilized tubulin was concentrated by glycerol dialysis and assembled. Cell bodies from the above disruption were detergent extracted and sonicated further to depolymerize the subpellicular microtubules. The solution was then cleared by centrifugation and concentrated by dialysis against glycerol. After clarification by centrifugation the dialyzate was taken through two cycles of assembly and disassembly. Assembly-competent tubulin was obtained from the cytoplasmic pool by using a column purification protocol. Cells were harvested, washed in buffer, and then lysed by gentle sonication. The cell bodies were removed by centrifugation and the supernatant run on a DEAE-Sephadex ion-exchange colomn. Tubulin was eluted using a salt cut of 0.55 M KC1, concentrated in an Amicon (Danvers, MA) concentrator, cleared by centrifugation, dialyzed at 4 °, and then polymerized at 37 ° for 30 min. Although this study developed the protocols for tubulin purification and polymerization the tubulin from all three fractions did not form complete microtubules. Rather, the tubulin polymerized into sheets and 3oD. G. Russell, D. Miller, and K. Gull, Mol. Cell Biol. 4, 779 (1984).
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ribbons. These protocols have since been used and adapted by Bramblett et a/. 3~,32to isolate putative MAPs. Stieger et al. 33 were able to partially purify the characterize tubulin from T. brucei by use of taxol-mediated assembly. Essentially, cells were lysed by sonication and centrifuged. Tubulin in the 100,000 g supernatant was assembled into microtubules by incubation at 30 ° in the presence of taxol and GTP. Recently, a general method has been developed which allows the complete purification of tubulin from T. brucei and its assembly in vitro into structurally normal microtubules.34 The technique involves the purification of tubulin by DEAE-Sephadex chromatography, Amicon concentration, glycerol dialysis, and then polymerization in vitro. The protocol is summarized below. 1. Cells grown to a density of 2 - 4 × 107/ml are harvested from 2 liters of medium by centrifugation at 1400 g for 5 min at 4 °. 2. Wash once in PEME supplemented with 4 M glycerol, 0.1 m M GTP, and 50/zg of leupeptin/ml, pH 6.9. 3. The washed cells are resuspended in 6 ml of the supplemented PEME, passed through a French press, and sonicated for four 30-sec periods, interspersed with 2-min incubations in an ice/water bath. 4. The sonicated cells are incubated on ice for 30 min, then centrifuged at 40,000 g for 30 min at 4 °. The supernatant is recentrifuged under the same conditions for 20 min. The resulting supernatant can be used immediately or stored at - 8 0 °. 5. The cell-free supernatant is applied at a flow rate of 25 ml/hr to a 28-mi DEAE-Sephadex column packed in a 30-ml disposable syringe, equilibrated with PEME containing 0.2 M KC1, 0.1 m M GTP, plus 12.5 /tg/ml of leupeptin. 6. The column is washed with the equilibration buffer and adherent protein is eluted with PEME containing 0.6 M KC1, 0.1 m M GTP, and 12.5/~g/ml leupeptin. Peak fractions are pooled and can be used immediately or stored at - 8 0 °. 7. The 0.6 M KC1 fraction from the column is concentrated to 2 - 3 ml in an Amicon ultrafiltration assembly. After filtration the preparation is further concentrated by dialysis overnight against 125 ml of PEME containing 8 M glycerol, 0.1 m M GTP, and 25/tg/ml leupeptin at 4* 3~G. T. Bramblett, S. C. Chang, and M. Flavin, Proc. Natl. Acad. Sci. U.S.A. 84, 3259 (1987). 32G. T. Bramblett, R. Kambadur, and M. Flavin, CellMot. Cytoskeleton 13, 145 (1989). 33j. Stieger, T. Wyler, and T. Seebeck, J. Biol. Chem. 259, 4596 (1984). T. H. MacRae and K. Gull, Biochem. J. (Tokyo) 265, 87 (1990).
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8. The dialyzed tubulin is centrifuged (40,000 g) twice (for 30 min and then 20 min) at 4 °, polymerized at 37 ° for 30 min in the presence of 10 m M Mg2+ and 1.8 m M GTP. The assembled tubulin is pelleted by centrifugation (40,000 g at 20 °) for 30 min. Since the microtubules pack poorly only 70% of the supernatant is removed and the loose pellet is resuspended in 50/A of PEME containing 1.8 m M GTP and incubated for 15 min at 37 °. 9. The preparation is centrifuged again at 40,000 g at 20°C. The supernatant is removed and the surface of the pellet washed with 100 #1 of PEME containing 0.1 m M G T P at 37 °. The pellet is resuspended in 200/zl of PEME containing 0.1 m M GTP and depolymerized by incubation on ice for 30 min, with gentle vortex mixing. 10. The suspension is then centrifuged at 40,000g for 30 min at 4°. The resulting supernatant containing the purified tubulin (final yield 0.9 mg) is stored at - 70 °. Trypanosome tubulin purified in the above manner assembles readily at a concentration of 2 mg/ml in the presence of 1.8 m M GTP at 37 °.
Microtubule-Associated Proteins in Trypanosomes The notable feature of the trypanosome cytoskeleton is the regularly spaced helical array of subpellicular microtubules. Ultrastructural studies have shown that the periodicity of these microtubules and their association with the plasma membrane is retained by a series of intermicrotubule cross-bridges and microtubule-membrane linkages. This has led many workers to search for the microtubule-associated proteins (MAPs) responsible for controlling such phenomena in trypanosomes. To date, a handful of candidate MAPs have been described. These putative MAPs are listed below together with a short description of their properties. A number of approaches have been taken to facilitate identification of subpellicular MAPs. Clearly, some of these have proved more effective than others and we will discuss the merits and disadvantages of the approaches adopted. The history of the identification of trypanosome MAPs illustrates the difficulties, yet necessity, of providing a precise definition of the term MAP. It is clear that some proteins identified as putative MAPs by their ability to associate with microtubules in vitro (in the detergent-extracted cytoskeleton or with reassembled microtubules) have turned out to be glycosomal enzymes. ~2,32These reports emphasize the requirement to work toward a more extensive definition of a putative MAP in terms of its behavior in vitro, its position within the intact cell, and preferably its molecular identity.
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MAP Fraction from Crithidia fasciculata A series of cytoplasmic MAPs were identified in C fasciculala. 31 These MAPs were obtained by purifying cytoplasmic microtubule protein as described earlier,3° and polymerizing stable microtubules in the presence of taxol and dimethyl sulfoxide. The MAPs were solubilized by extraction of the pellet with 0.6 M sodium chloride buffer while the microtubules were stabilized with taxol. After concentration by centricon filter and dialysis, several proteins were present in the enhanced fraction. The most notable proteins in this fraction possessed molecular weights of 130K, 90K, 65K, 49K, 36K, and 33K. Incubation of this fraction with tubulin induced microtubule polymerization, although it was 10 times less effective than the MAP fraction isolated from mammalian brain. Two of the trypanosome proteins, MAP33 and MAP40, were concentrated in the polymer fraction. These authors 31,32also isolated a set of three proteins (corset proteins, COPs) which were associated with the microtubule corset of C. fasciculata. Essentially pellicular microtubular protein was isolated from this organism as described earlier, 3° induced to assemble by dialysis against glycerol, and the microtubuIes discarded. The remaining fraction contained the COPs. The proteins were then fractionated by salt gradient elution from a cationexchange Mono S column. The three proteins isolated by this method were termed COP41, COP61, and COP33 (the numbers referring to their apparent molecular weights). COP41. This protein, when incubated with polymerized brain tubulin, formed cross-links which were periodic along the lengths of the microtubules. Also, the native protein appeared to be tetrameric with a molecular weight of approximately 160K. However, immunogold localization of this protein in C. fasciculata revealed that an antibody specific for this protein bound only to glycosomes. Upon further investigation COP41 proved to be glyceraldehyde 3-P-dehydrogenase (GAPDH). Thus, in cells this enzyme was confined in glycosomes and only bound to corset microtubules after release by homogenization. COP61. This protein, which forms a dimer of Mr 120K, bound avidly .to preassembled microtubules but did not bundle or cross-link them. Immunogold localization with a polyclona! antibody to COP61 suggested that the antibody showed some affinity for cytoskeletons when incubated with them as whole,mounts, but no clear affinity for microtubules in intact cells. COP33. This protein readily formed cross-links between preassembled
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microtubules and is thought to form a tetramer of Mr 135K. The crosslinks between the microtubules had a periodicity of 8.5 nm. However, a polyclonal antibody raised to COP33 showed no specific binding to cytoskeletons or cell sections. Thus, again the identification of this protein as a MAP requires confirmation. MAPs from Trypanosoma brucei The search for trypanosomal MAPs has also produced some candidates from T. brucei. p60. A protein of M, 60K was isolated from T. brucei by Seebeck et al. 35 The protein was purified by a succession of gel-filtration and ion-exchange chromatography or by velocity sedimentation in a glycerol gradient from a crude cytoskeletal extract. 1360was found to (1) copolymerize with tubulin, (2) bind to preassembled microtubules, (3) bind to synthetic liposomes, and (4) cross-link microtubules and membrane vesicles. All this seemed to suggest that p60 was a trypanosomal MAP. However, it now appears that p60 is in fact a glycosomal protein, phosphoenolpyruvate carboxykinase. 12.36 p41. This protein was isolated from T. brucei cytoskeletons by extraction with 0.1% Triton X-100 and 1 m M EGTA. 37 The protein, once isolated, was found to possess covalently bound fatty acid. 1341 was found to remain tightly bound to the cytoskeleton if calcium ions were present, but it could also be selectively released if cytoskeletons were incubated in excess EGTA. Purified 1341 was found to bind to isolated cytoskeletons and to preassembled microtubules of trypanosome tubulin. p320. p320 is a heat-stable protein which was isolated from the pellicular tubulin fraction of T. brucei by heat treatment of the 0.75 M sodium chloride-solubilized fraction of the cytoskeleton.36 It was localized to the pellicular microtubules of T. brucei by immunogold probing with a specific polyclonal antibody, p320 also polymerized with tubulin when trypanosome cell lysates were induced to assemble by taxol. A cloned segment of the p320 gene indicated that the predicted protein sequence consisted of 50 nearly identical tandem repeats of 38 amino acids. To date this remains the most promising and best described candidate for a trypanosomal MAP. 52K Protein. Solubilization of the subpellicular microtubules of T. brucei by a high-strength salt solution, and further fractionation by Mono S
3s T. Seebeck, V. Kueng, T. Wyler, and M. Muller, Proc. Natl, Acad. Sci. U.S.A. 85, 1101 (1988). 36A. Schneider, A. Hemphill, T. Wyler, and A. Seebeck, Science 241, 459 (1988). 37A. Schneider, W. Eichenberger, and T. Seebeck, J. Biol. Chem. 263, 6472 (1988).
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cation-exchange column chromatography, led to an Mr 52K protein being eluted. 3a The 52K protein, when bound to nitrocellulose, gave an indication of tubulin binding. Also, when this protein was incubated with brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular cross-links between the microtubules. However, there is no biochemical or immunological characterization of this protein, nor information as to its location in the intact trypanosome cell. Again, therefore, its identification as a defined MAP must await further validation. We now possess a rather extensive knowledge of the structural components and ultrastructural organization of the trypanosome cytoskeleton. We also have a good understanding of the molecular biology and cell biology of the major structural protein, tubulin. The precise shape and form of this cell makes it an extremely suitable system in which to study the assembly of cytoskeletal structures possessing very high spatial order. The regulatory, interacting proteins responsible for the assembly maintenance, and functional-properties of the cytoskeleton are likely to be of great general interest. We have only just started to gain an insight into the identity of these proteins and it is clear that our best understanding of their roles will come from multifaceted studies which include molecular, cellular, and biochemical approaches. Acknowledgments Work in this laboratoryreceivedfinancialsupport from the UNDP/WorldBank/WHO special program for Researchand Training in Tropical Diseases. D.R.R. was funded by a SERC studentshipand P.B. was fundedby a WellcomePrizestudentship.
3s N. Balaban, H. K. Waithaka, A. R. Njogu, and R. Goldman. Cell Mot. Cytoskeleton 14, 393 (1989).
