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SDS/polyacrylamide-gel electrophoresis of trypanosome tubulin during purification Fractions from different stages of the trypanosome tubulin purification procedure were electrophoresed on onedimensional SDS/polyacrylamide gels and stained with Coomassie Blue R250. The lanes contained: 1, cell-free supernatant (80,ug); 2, 0.6 M-KCI DEAE fraction after concentration against an Amicon Diaflow PM10 membrane and dialysis against PEME containing 8 M-glycerol (32,ug); 3, purified (cycled) trypanosome tubulin (10 tg); 4, sheep brain microtubule protein prepared by three cycles of assembly/disassembly (8 ,tg); 5, purified (cycled) trypanosome tubulin (40 ,ug). Abbreviation: TUB, tubulin.
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Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2...
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... assembly assay Purified trypanosome tubulin in PEME was incubated with 1.8 mM-GTP at 37 'C for 30 min at a final con- centration of 2 mg/ml. Where indicated, glycerol was added to 4 M and Mg2" to 10 mm. Microtubule-reactive drugs used in assembly assays were prepared as stock solutions in PEME (colcemid and vinblastine), dimethyl sulphoxide diluted to 20 % with PEME (maytansine and taxol) or ethanol diluted to 500 with PEME (nocodazole). The concentrations of the stock solutions were such that a 1:10 (v/v) dilution in the assembly mixture resulted in the final drug concentrations indicated in Table 2. Stock solutions were used immediately after preparation and then discarded. Controls, consisting of solvents in the absence of drugs, were tested. Electron microscopy Samples for electron microscopy were fixed by diluting 1: 1 in PEME containing 40 (Fig. 1, lane 1). The major band interacted with anti-tubulin antibodies after blot- ting to nitrocellulose (results not shown). We were unable to polymerize the tubulin in the cell-free extracts unless taxol, at 20 /M, was added, resulting in the assembly of short, ill-formed, microtubules (Fig. 2a). The taxol was kindly given by Dr. Matthew Suffness, Natural Products Branch, NCI, Bethesda, MD, U.S.A. Initial attempts to purify the tubulin by use of phosphocellulose P-Il chromatography and (NH4)2SO4 precipitation proved unsuccessful, mainly owing to difficulty in resolubilizing the (NH4)2SO4 precipitate. Chromatography on DEAE- Sephadex followed by concentration against an Amicon Diaflow PM1O membrane and dialysis in PEME con- taining 8 M-glycerol yielded a protein solution in which the tubulin assembled into long, often poorly formed, microtubules on addition of GTP (Fig. 2b). Addition of Mg2+ at 10 mm had little apparent effect on microtubule ultrastructure at this stage of purification (results not shown). On SDS/polyacrylamide gels, the Amicon- concentrated 0.6 M-KCI fraction from DEAE-Sephadex exhibited a major band of tubulin and several con- taminating polypeptides (Fig. 1, lane 2). Electrophoresis of the material obtained after assembly/disassembly of the Amicon-concentrated DEAE fraction revealed a single band when 10-20 ,ug of protein was applied to the gel (Fig. 1, lane 3 t The microtubules were poorly formed and the amount of assembly appeared to be decreased when colcemid was ...
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... assembly assay Purified trypanosome tubulin in PEME was incubated with 1.8 mM-GTP at 37 'C for 30 min at a final con- centration of 2 mg/ml. Where indicated, glycerol was added to 4 M and Mg2" to 10 mm. Microtubule-reactive drugs used in assembly assays were prepared as stock solutions in PEME (colcemid and vinblastine), dimethyl sulphoxide diluted to 20 % with PEME (maytansine and taxol) or ethanol diluted to 500 with PEME (nocodazole). The concentrations of the stock solutions were such that a 1:10 (v/v) dilution in the assembly mixture resulted in the final drug concentrations indicated in Table 2. Stock solutions were used immediately after preparation and then discarded. Controls, consisting of solvents in the absence of drugs, were tested. Electron microscopy Samples for electron microscopy were fixed by diluting 1: 1 in PEME containing 40 (Fig. 1, lane 1). The major band interacted with anti-tubulin antibodies after blot- ting to nitrocellulose (results not shown). We were unable to polymerize the tubulin in the cell-free extracts unless taxol, at 20 /M, was added, resulting in the assembly of short, ill-formed, microtubules (Fig. 2a). The taxol was kindly given by Dr. Matthew Suffness, Natural Products Branch, NCI, Bethesda, MD, U.S.A. Initial attempts to purify the tubulin by use of phosphocellulose P-Il chromatography and (NH4)2SO4 precipitation proved unsuccessful, mainly owing to difficulty in resolubilizing the (NH4)2SO4 precipitate. Chromatography on DEAE- Sephadex followed by concentration against an Amicon Diaflow PM1O membrane and dialysis in PEME con- taining 8 M-glycerol yielded a protein solution in which the tubulin assembled into long, often poorly formed, microtubules on addition of GTP (Fig. 2b). Addition of Mg2+ at 10 mm had little apparent effect on microtubule ultrastructure at this stage of purification (results not shown). On SDS/polyacrylamide gels, the Amicon- concentrated 0.6 M-KCI fraction from DEAE-Sephadex exhibited a major band of tubulin and several con- taminating polypeptides (Fig. 1, lane 2). Electrophoresis of the material obtained after assembly/disassembly of the Amicon-concentrated DEAE fraction revealed a single band when 10-20 ,ug of protein was applied to the gel (Fig. 1, lane 3 t The microtubules were poorly formed and the amount of assembly appeared to be decreased when colcemid was ...
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... assembly assay Purified trypanosome tubulin in PEME was incubated with 1.8 mM-GTP at 37 'C for 30 min at a final con- centration of 2 mg/ml. Where indicated, glycerol was added to 4 M and Mg2" to 10 mm. Microtubule-reactive drugs used in assembly assays were prepared as stock solutions in PEME (colcemid and vinblastine), dimethyl sulphoxide diluted to 20 % with PEME (maytansine and taxol) or ethanol diluted to 500 with PEME (nocodazole). The concentrations of the stock solutions were such that a 1:10 (v/v) dilution in the assembly mixture resulted in the final drug concentrations indicated in Table 2. Stock solutions were used immediately after preparation and then discarded. Controls, consisting of solvents in the absence of drugs, were tested. Electron microscopy Samples for electron microscopy were fixed by diluting 1: 1 in PEME containing 40 (Fig. 1, lane 1). The major band interacted with anti-tubulin antibodies after blot- ting to nitrocellulose (results not shown). We were unable to polymerize the tubulin in the cell-free extracts unless taxol, at 20 /M, was added, resulting in the assembly of short, ill-formed, microtubules (Fig. 2a). The taxol was kindly given by Dr. Matthew Suffness, Natural Products Branch, NCI, Bethesda, MD, U.S.A. Initial attempts to purify the tubulin by use of phosphocellulose P-Il chromatography and (NH4)2SO4 precipitation proved unsuccessful, mainly owing to difficulty in resolubilizing the (NH4)2SO4 precipitate. Chromatography on DEAE- Sephadex followed by concentration against an Amicon Diaflow PM1O membrane and dialysis in PEME con- taining 8 M-glycerol yielded a protein solution in which the tubulin assembled into long, often poorly formed, microtubules on addition of GTP (Fig. 2b). Addition of Mg2+ at 10 mm had little apparent effect on microtubule ultrastructure at this stage of purification (results not shown). On SDS/polyacrylamide gels, the Amicon- concentrated 0.6 M-KCI fraction from DEAE-Sephadex exhibited a major band of tubulin and several con- taminating polypeptides (Fig. 1, lane 2). Electrophoresis of the material obtained after assembly/disassembly of the Amicon-concentrated DEAE fraction revealed a single band when 10-20 ,ug of protein was applied to the gel (Fig. 1, lane 3 t The microtubules were poorly formed and the amount of assembly appeared to be decreased when colcemid was ...
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... The microtubules tended to form ribbons, but this also occurred in the presence of 5 % ethanol, the final concentration of the nocodazole solvent within assembly reactions. very lightly staining bands, all of lower molecular mass than tubulin, were visible (Fig. 1, lane 5). Whether these bands are contaminating proteins or breakdown products of the tubulin is uncertain. Protein yields during purification are summarized in Table ...
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... one-dimensional SDS/polyacrylamide gels, the a- and ,8-tubulins were either not resolved (Fig. 1, lane 3) or resolved very poorly (results not shown), whereas on the same gels sheep brain tubulin separated clearly into two subunits (Fig. 1, lane 4). General monoclonal antibodies to a-and ,8-tubulin, as well as those that specifically recognize the acetylated and tyrosinated forms of a- tubulin, all reacted well with purified trypanosome tubulin on Western blots (Fig. ...
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... one-dimensional SDS/polyacrylamide gels, the a- and ,8-tubulins were either not resolved (Fig. 1, lane 3) or resolved very poorly (results not shown), whereas on the same gels sheep brain tubulin separated clearly into two subunits (Fig. 1, lane 4). General monoclonal antibodies to a-and ,8-tubulin, as well as those that specifically recognize the acetylated and tyrosinated forms of a- tubulin, all reacted well with purified trypanosome tubulin on Western blots (Fig. ...
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... Just like paclitaxel and related MT-stabilizing agents, MT-depolymerizing vinca alkaloids like vinblastine (60) and vincristine (61, Figure 19), which interact with a binding site located within the β-tubulin subunit at the dimer interface [195,196], have also been reported as potent antiprotozoal agents [197]. The assembly of purified trypanosome tubulin was inhibited in cell-free assays by vinblastine (40 μM) and other natural products that are known to interact with the vinca binding site, such as maytansine (62, Figure 19) [198]. Additional studies with purified Leishmania tubulin confirmed that low micromolar concentrations of all of the vinca domain agents tested, including ansamitocin P3, rhizoxin, and hemiasterlin (63-65, Figure 19), effectively inhibited tubulin assembly [188]. ...
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... Tubulin has been purified from Crithidia, Leishmania, and T. brucei [72,73,120]; interestingly, this tubulin has a tendency to form ribbon and sheet-like structures instead of tubules. Tubulin purified from procyclic T. brucei polymerizes into tubes in the presence of high magnesium and GTP concentrations. ...
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... In contrast to the microtubules of mammalian cells, SPMT are resistant to low temperatures and drugs that usually promote microtubule depolymerization [10]; thus, SPMT contribute to the stabilization of the trypanosomatids shape. Transversal ultrathin sections of Leptomonas samueli fixed with glutaraldehyde and tannic acid showed that trypanosomatids' SPMT are formed by 13 typical protofilaments [11]. ...
... In the light of conventional kinetochore research, it will be important to invest efforts to obtain recombinant KKT proteins and perform microtubule co-sedimentation assays. Although tubulins purified from bovine brain are commonly used in sedimentation assays, it may be important to use trypanosome tubulins to detect binding [165,166]. ...
The kinetochore is the macromolecular protein complex that drives chromosome segregation in eukaryotes. Its most fundamental function is to connect centromeric DNA to dynamic spindle microtubules. Studies in popular model eukaryotes have shown that centromere protein (CENP)-A is critical for DNA-binding, whereas the Ndc80 complex is essential for microtubule-binding. Given their conservation in diverse eukaryotes, it was widely believed that all eukaryotes would utilize these components to make up a core of the kinetochore. However, a recent study identified an unconventional type of kinetochore in evolutionarily distant kinetoplastid species, showing that chromosome segregation can be achieved using a distinct set of proteins. Here, I review the discovery of the two kinetochore systems and discuss how their studies contribute to a better understanding of the eukaryotic chromosome segregation machinery.
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Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the beta-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the
crystallographic structure of the bovine protein as template.We identified mutations on the Leishmania beta-tubulin gene sequences on regions related to the putative colchicine-binding pocket,which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The samemutations were found in the beta-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational
changes include an elongation and torsion of an 𝛼-helix structure and displacement to the inside of the pocket of one beta-sheet that hinders access of colchicine.We propose that kinetoplastid organisms showresistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.
... The vinca alkaloid agents, such as vinblastine, exhibited a high effect on Leishmania and other kinetoplastids [15]. The isolation and purification of tubulin from kinetoplastids such as T. brucei [16] and Leishmania spp. [14,17] have been carried out successfully, and evaluation of such preparations demonstrated their suitability for use in screening of drugs with selective antimitotic activity, such as dinitroaniline herbicides, oryzalin, and derivatives compounds [14,18]. ...
Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the β -tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania β -tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the β -tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α -helix structure and displacement to the inside of the pocket of one β -sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.
... Similar to the native T. brucei tubulin isoforms (31), the molecular weights (MW) for the bacterially expressed tubulins were identical (∼50kDa) and indistinguishable by gel migration. Furthermore, from sequence data of the cloned genes, the predicted MW for these proteins were also nearly identical (∼49673.0195Da vs. 49448.6367Da, ...
There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth.
T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450µg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera.
Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions.
Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes.
... The MT arrays of trypanosomes are highly resistant to depolymerization upon cold treatment [27,28] and have a low sensitivity to nocodazole [29,30]. In T. brucei, several MAPs have been characterized, but notably none have been described as being flagellar specific [20,31,32,33,34,35,36,37]. ...
... To study TbSAXO MT stabilizing functions directly, and to circumvent the fact that the trypanosome cytoskeleton is unusually stable [20,27,29,30], we expressed a recombinant TbSAXO-Myc protein in the U-2 OS human cell line (human bone osteosarcoma epithelial cells) [59]. As with many mammalian cell lines, when U-2 OS cells in culture are incubated either at 4uC or in the presence of nocodazole, interphase MTs depolymerize (Fig. S1A, B). ...
In vertebrates the microtubule-associated proteins MAP6 and MAP6d1 stabilize cold-resistant microtubules. Cilia and flagella have cold-stable microtubules but MAP6 proteins have not been identified in these organelles. Here, we describe TbSAXO as the first MAP6-related protein to be identified in a protozoan, Trypanosoma brucei. Using a heterologous expression system, we show that TbSAXO is a microtubule stabilizing protein. Furthermore we identify the domains of the protein responsible for microtubule binding and stabilizing and show that they share homologies with the microtubule-stabilizing Mn domains of the MAP6 proteins. We demonstrate, in the flagellated parasite, that TbSAXO is an axonemal protein that plays a role in flagellum motility. Lastly we provide evidence that TbSAXO belongs to a group of MAP6-related proteins (SAXO proteins) present only in ciliated or flagellated organisms ranging from protozoa to mammals. We discuss the potential roles of the SAXO proteins in cilia and flagella function.
... Nevertheless, some differences have been noted over the years indicating that tubulin isotype composition may determine microtubule dynamics, protein association to the microtubule lattice, and drugbinding parameters (Banerjee et al., 1990;Derry et al., 1997;Lu and Luduena, 1994;Newton et al., 2002;Panda et al., 1994). Therefore, it is useful to analyze the content in tubulin preparations from nonneuronal tissues and cells in culture (Bellocq et al., 1992;Farrell, 1982;Fourest-Lieuvin, 2006;Kilmartin, 1981;MacRae and Gull, 1990;Macrae and Luduena, 1984;Maekawa and Sakai, 1978;Morejohn and Fosket, 1982;Murphy, 1991;Rudiger and Weber, 1993;Verdier-Pinard et al., 2009;Weatherbee et al., 1980). This analysis may help investigators in refining the interpretation of the data from their in vitro assays. ...
New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.
... Several compounds have been described as microtubule-interacting agents, based on their ability to induce or inhibit the tubulin assembly in microtubules. Even though a high degree of tubulin amino acid conservation is found throughout the evolution, there are some reports suggesting that colchicine and benzimidazoles, two classical mammalian tubulin inhibitors, had only a slight effect on the trypanosomatids tubulin polymerization [38, 39]. On the other hand, taxol, a microtubule network stabilizing agent, affects the parasite cytoskeleton in a dose-dependent manner [40]. ...
Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD(+)-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD(+)-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of alpha-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of alpha-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD(+)-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.
