Trypanosoma brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.     

Two related subpellicular cytoskeleton-associated proteins in Trypanosoma brucei stabilize microtubules

The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed in T. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis.     

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.     

Proteomics and the Trypanosoma brucei cytoskeleton: advances and opportunities

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.     

Isolation of a subpellicular microtubule protein from Trypanosoma brucei that mediates crosslinking of microtubules

The cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS-gel electrophoresis). Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose-bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubules. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.     

A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form.

Cell differentiation in Trypanosoma brucei involves highly regulated changes in morphology, proliferation and metabolism. However, the controls of these developmental processes are unknown. We have identified two novel proteins from the rare CCCH zinc finger family, each <140 amino acids in length and implicated in life cycle regulation. TbZFP1 is transiently enriched during differentiation from the bloodstream to procyclic form, whereas tbZFP2, when ablated in bloodstream forms by RNA interference, inhibits this developmental step. Moreover, expressing an ectopic copy of tbZFP2 results in a dramatic procyclic stage-specific remodelling of the trypanosome cytoskeleton similar to the morphogenic events of differentiation. This phenotype, we term 'nozzle', involves polar extension of microtubules at the posterior end of the cell and is dependent upon a motif hitherto restricted to E3 ubiquitin ligases. TbZFP1 and tbZFP2 represent the first molecules implicated in the control of trypanosome differentiation to the procyclic form.     

JVG9, a benzimidazole derivative,alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.     

Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55-kDa subunits, alpha- and beta-tubulin; in Trypanosoma brucei, the tubulin-coding sequences are clustered in a 40-kb fragment of tandemly repeated alpha- and beta-tubulin genes separated by a 170-bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5' 35-nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post-translationally modified in 2 ways: the C-terminal tyrosine of alpha-tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin-tyrosine ligase; alpha-tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the beta-tubulin form defined by the monoclonal antibody 1B41 is sequestered into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so-called "subpellicular microtubule quartet" (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.     

Detection of the microtubule cytoskeleton of the coccidian Toxoplasma gondii and the hemoflagellate Leishmania donovani by monoclonal antibodies specific for beta-tubulin

Seven monoclonal antibodies specific for mammalian beta-tubulin demonstrate the microtubule cytoskeleton of Toxoplasma gondii and Leishmania donovani by indirect immunofluorescence microscopy. Immunoblots of T. gondii and L. donovani proteins separated by SDS polyacrylamide gel electrophoresis confirm the specificity of the monoclonal antibodies for tubulin. Differential staining of flagellar and subpellicular microtubule populations was not seen in L. donovani with these antibodies. All seven antibodies also detected the subpellicular microtubules of T. gondii, but the polar ring and conoid of this organism was not visualized by any of them. This technique provides a rapid and specific way to assess microtubular organization in whole organisms.     

Host-parasite interactions and trypanosome morphogenesis: a flagellar pocketful of goodies

Trypanosomes are characterised by the possession of a single flagellum and a subpellicular microtubule cytoskeleton. The flagellum is more than an organelle for motility; its position and polarity along with the sub-pellicular cytoskeleton enables the morphogenesis of a distinct flagellar pocket and the flagellar basal body is responsible for positioning and segregating the kinetoplast--the mitochondrial genome. Recent work has highlighted the molecules and morphogenesis of these cytoskeletal/flagellum structures and how dynamic events, occurring in the flagellar pocket and kinetoplast, are critical for host-parasite interactions.     

Detection of the Microtubule Cytoskeleton of the Coccidian Toxoplasma gondii and the Hemoflagellate Leishmania donovani by Monoclonal Antibodies Specific for β-Tubulin1

Seven monoclonal antibodies specific for mammalian β-tubulin demonstrate the microtubule cytoskeleton of Toxoplasma gondii and Leishmania donovani by indirect immunofluorescence microscopy. Immunoblots of T. gondii and L. donovani proteins separated by SDS polyacrylamide gel electrophoresis confirm the specificity of the monoclonal antibodies for tubulin. Differential staining of flagellar and subpellicular microtubule populations was not seen in L. donovani with these antibodies. All seven antibodies also detected the subpellicular microtubules of T. gondii, but the polar ring and conoid of this organism was not visualized by any of them. This technique provides a rapid and specific way to assess microtubular organization in whole organisms.     

Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.     

Repression of polymerase I-mediated gene expression at Trypanosoma brucei telomeres

The African trypanosome, Trypanosoma brucei, is a flagellated pathogenic protozoan that branched early from the eukaryotic lineage. Unusually, it uses RNA polymerase I (Pol I) for mono-telomeric expression of variant surface glycoprotein (VSG) genes in bloodstream-form cells. Many other subtelomeric VSG genes are reversibly repressed, but no repressive DNA sequence has been identified in any trypanosomatid. Here, we show that artificially seeded de novo telomeres repress Pol I-dependent gene expression in mammalian bloodstream and insect life-cycle stages of T. brucei. In a telomeric VSG expression site, repression spreads further along the chromosome and this effect is specific to the bloodstream stage. We also show that de novo telomere extension is telomerase dependent and that the rate of extension correlates with the expression level of the adjacent gene. Our results show constitutive telomeric repression in T. brucei and indicate that an enhanced, developmental stage-specific repression mechanism controls antigenic variation.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.     

Microtubule plus-end tracking proteins in differentiated mammalian cells

Differentiated mammalian cells are often characterized by highly specialized and polarized structure. Its formation and maintenance depends on cytoskeletal components, among which microtubules play an important role. The shape and dynamic properties of microtubule networks are controlled by multiple microtubule-associated factors. These include molecular motors and non-motor proteins, some of which accumulate specifically at the growing microtubule plus-ends (the so-called microtubule plus-end tracking proteins). Plus-end tracking proteins can contribute to the regulation of microtubule dynamics, mediate the cross-talk between microtubule ends, the actin cytoskeleton and the cell cortex, and participate in transport and positioning of structural and regulatory factors and membrane organelles. Malfunction of these proteins results in various human diseases including some forms of cancer, neurodevelopmental disorders and mental retardation. In this article we discuss recent data on microtubule dynamics and activities of microtubule plus-end binding proteins important for the physiology and pathology of differentiated mammalian cells such as neurons, polarized epithelia, muscle and sperm cells.     

Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner

Leishmania parasites experience variable nutrient levels as they cycle between the extracellular promastigote stage in the sandfly vector and the obligate intracellular amastigote stage in the mammalian host. Here we show that the surface expression of three Leishmania mexicana hexose and myo-inositol transporters is regulated in both a stage-specific and nutrient-dependent manner. GFP-chimeras of functionally active hexose transporters, LmGT2 and LmGT3, and the myo-inositol transporter, MIT, were primarily expressed in the cell body plasma membrane in rapidly dividing promastigote stages. However MIT-GFP was mostly rerouted to the multivesicular tubule (MVT)-lysosome when promastigotes reached stationary phase growth and all three nutrient transporters were targeted to the amastigote lysosome following transformation to in vitro differentiated or in vivo imaged amastigote stages. This stage-specific decrease in surface expression of GFP-tagged transporters correlated with decreased hexose or myo-inositol uptake in stationary phase promastigotes and amastigotes. The MVT-lysosme targeting of the MIT-GFP protein was reversed when promastigotes were deprived of myo-inositol, indicating that nutrient signals can override stage-specific changes in transporter distribution. The surface expression of the hexose and myo-inositol transporters was not regulated by interactions with the subpellicular cytoskeleton, as both classes of transporters associated with detergent-resistant membranes. LmGT3-GFP and MIT-GFP proteins C-terminally modified with mono-ubiquitin were constitutively transported to the MVT-lysosome, suggesting that ubiquitination may play a key role in regulating the subcellular distribution of these transporters and parasite adaptation to different nutrient conditions.     

Regulation of Focal Adhesion Dynamics and Cell Motility by the EB2 and Hax1 Protein Complex

Cell migration is a fundamental cellular process requiring integrated activities of the cytoskeleton, membrane, and cell/extracellular matrix adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2, and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe, whereas, in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about the cellular function of EB2. By quantitative proteomics, we identified mammalian HCLS1-associated protein X-1 (HAX1) as an EB2-specific interacting protein. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover require interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.     

Glycoproteins of trypanosomes: their biosynthesis and biological significance

1. Trypanosomes are unicellular parasites that cause human sleeping sickness in Africa and Chagas' disease in South America. Glycoproteins are important components of their plasma membrane. 2. The bloodstream form of the extracellular salivarian African trypanosome (e.g. Trypanosoma brucei) has the ability to express on its cell surface a repertoire of variant surface glycoproteins (VSGs) and in so doing, evades the immune response of the host (antigenic variation). 3. The VSG is probably synthesized initially in a manner like that of the membrane-bound glycoproteins of mammalian systems, but it also undergoes some novel post-translational modifications. 4. The stercorarian South American trypanosome (Trypanosoma cruzi) is an intracellular parasite which expresses different glycoproteins on its plasma membrane at various stages of its life-cycle, but does not exhibit antigenic variation. 5. The biosynthesis and functions of trypanosomal glycoproteins are compared with those of mammalian glycoproteins, and are discussed with particular reference to potential targets for chemotherapy and immunotherapy of trypanosomiasis.