Biomedical subjects
Publications and source records attributed to K Gull.
Amongst the earliest eukaryotes, trypanosomes have developed conventional organelles but sometimes with extreme features rarely seen in other organisms. This is the case of the flagellum, containing conventional and unique structures whose role in infectivity is still enigmatic.
The genome projects represent one of the most important developments in our knowledge of parasites. However, translation of this knowledge into an understanding of parasite biology and then on to drugs, vaccines and other healthcare developments for the diseases will need some élan and clarity of thought by scientists and funding organizations. Only then will the activity associated with post-genomics be turned from what I have termed 'genome babble' to real opportunities in understanding these parasites.
The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread applicability in studies of gene function and cellular processes in parasites.
In this article, Klaus Ersfeld, Sara Melville and Keith Gull review current understanding of the structural organization of the nucleus of Trypanosoma brucei, and summarize recent data pertinent to the organization of its genome. Until recently, the cell biology of the trypanosome nucleus and issues of DNA organization and gene expression have often been treated as separate themes. However, recent work emphasizes the need for a more holistic approach to understanding these aspects of the biology of this parasite.
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The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.
Species of the trypanosomatid parasite genera Trypanosoma and Leishmania exhibit a particular range of cell shapes that are defined by their internal cytoskeletons. The cytoskeleton is characterized by a subpellicular corset of microtubules that are cross-linked to each other and to the plasma membrane. Trypanosomatid cells possess an extremely precise organization of microtubules and filaments, with some of their organelles, such as the mitochondria, kinetoplasts, basal bodies, and flagella, present as single copies in each cell. The duplication of these structures and changes in their position during life cycle differentiations provide markers and insight into events involved in determining cell form and division. We have a rapidly increasing catalog of these structures, their molecular cytology, and their ontogeny. The current sophistication of available molecular genetic techniques for use in these organisms has allowed a new functional analysis of the cytoskeleton, including functions that are intrinsic to the proliferation and pathogenicity of these parasites.
The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.
The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.
The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or 'blob'. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.
Trypanosoma brucei has a single nucleus and a single kinetoplast (the mitochondrial genome). Each of these organelles has a distinct S phase, which is followed by a segregation period, prior to cell division. The segregation of the two genomes takes place in a specific temporal order by interaction with microtubule-based structures, the spindle for nuclear DNA and the flagellum basal bodies for the kinetoplast DNA. We used rhizoxin, the anti-microtubule agent and polymerisation inhibitor, or the nuclear DNA synthesis inhibitor aphidicolin, to interfere with cell cycle events in order to study how such events are co-ordinated. We show that T. brucei cytokinesis is not dependent upon either mitosis or nuclear DNA synthesis, suggesting that there are novel cell cycle checkpoints in this organism. Moreover, use of monoclonal antibodies to reveal cytoplasmic events such as basal body duplication shows that some aphidicolin treated cells appear to be in G(1) phase (1K1N) but have activated some cytoplasmic events characteristic of G(2) phase (basal body segregation). We discuss a possible dominant role in trypanosomes for kinetoplast/basal body segregation in control of later cell cycle events such as cytokinesis
Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the alpha-tubulin mRNA 5' untranslated region (5' UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic alpha-tubulin 5' UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of alpha-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to alpha-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.
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Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5A and -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3'-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence.
Trypanosoma brucei undergoes dramatic stage-specific changes in surface antigen expression, metabolic development, cellular morphogenesis and cell-cycle control. These events can be studied in detail during the transition between the bloodstream stumpy stage and the tsetse fly midgut procyclic form. This differentiation can be induced in vitro, is synchronous in the population and there are abundant markers for stage-regulated and differentiation events. We have used this differentiation system to investigate the role of de novo transcription during different phases of this well-characterised cellular transformation. Our experiments implicate early transcriptional involvement in shedding of the variable surface glycoprotein coat, cell restructuring and cell-cycle re-entry. The synchrony of differentiation has also been exploited to identify transcripts which define distinct regulated processes during this differentiation. The transcripts identified provide good coverage of the different molecular regulation events that accompany this life-cycle transformation. These included a surface antigen gene (encoding procyclin/PARP), a cell cycle regulated component (encoding histone H2B), a homologue of the Leishmania activated protein kinase C receptor (LACK) and a putative gene for sub unit VI of cytochrome c oxidase.
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We achieved the direct visualisation of gene organisation in Trypanosoma brucei using fluorescent in situ hybridisation on extended nuclear DNA fibres. We demonstrated the repetitive nature of the tubulin gene cluster, which consists of up to 19 alpha- and beta-tubulin genes arranged in tandem repeats.