Characterization of the single end-binding protein homologue TbEB1 in Trypanosoma brucei

Trypanosoma brucei is an early branching protist whose cytoskeleton is predominantly composed of microtubules. The cytoskeleton is the key component involved in cellular shape, organelle positioning, and cytokinesis. T. brucei possesses one homologue of the end-binding protein (EB) 1 family: TbEB1. EB1 proteins belong to the highly conserved family of plusend tracking proteins (+TIPs) and are master regulators of +TIP networks, thereby coordinating microtubule dynamics. In trypanosomes, the TbEB1 labeling pattern varies throughout the cell cycle at the posterior cell pole in immunofluorescence experiments. Additionally, TbEB1 localized to the FAZ/ FAZ region and the subpellicular microtubule array. Depletion of TbEB1 resulted in a severe growth defect, accumulation of atypical cell cycle stages, and an impediment of cytokinesis. Over-expression of TbEB1 resulted in similar phenotypes, however, less severe. Ectopic expression in a mammalian cell line pointed towards an evolutionary conserved mode of action. Altogether this suggests a role of TbEB1 in microtubule polymerization and indicates its putative involvement in cytokinesis

Cold storage and cryopreservation of tick cell lines

<p>Abstract</p> <p>Background</p> <p>Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from <it>Rhipicephalus </it>(<it>Boophilus) decoloratus</it>, <it>Rhipicephalus </it>(<it>Boophilus) microplus, Ixodes ricinus </it>and <it>Ixodes scapularis</it>. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.</p> <p>Results</p> <p>Cold storage at 6°C for up to 30 days was successful in preserving <it>R</it>. (<it>B.) microplus</it>, <it>R</it>. (<it>B.) decoloratus, I. ricinus </it>and <it>I. scapularis </it>cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the <it>R</it>. (<it>B</it>.) <it>decoloratus </it>cells did not survive freezing in SPG and of the other three species, only the <it>R</it>. (<it>B</it>.) <it>microplus </it>cells resumed growth during the observation period.</p> <p>Conclusions</p> <p>This constitutes the first report on successful short-term refrigeration of cells derived from <it>R</it>. (<it>B.) decoloratus</it>, <it>R</it>. (<it>B.) microplus</it>, and <it>I. ricinus</it>, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.</p

Characterization of the Single End-Binding Protein Homologue TbEB1 in Trypanosoma brucei

Trypanosoma brucei is an early branching protist whose cytoskeleton is predominantly composed of microtubules. The cytoskeleton is the key component involved in cellular shape, organelle positioning, and cytokinesis. T. brucei possesses one homologue of the end-binding protein (EB) 1 family: TbEB1. EB1 proteins belong to the highly conserved family of plusend tracking proteins (+TIPs) and are master regulators of +TIP networks, thereby coordinating microtubule dynamics. In trypanosomes, the TbEB1 labeling pattern varies throughout the cell cycle at the posterior cell pole in immunofluorescence experiments. Additionally, TbEB1 localized to the FAZ/ FAZ region and the subpellicular microtubule array. Depletion of TbEB1 resulted in a severe growth defect, accumulation of atypical cell cycle stages, and an impediment of cytokinesis. Over-expression of TbEB1 resulted in similar phenotypes, however, less severe. Ectopic expression in a mammalian cell line pointed towards an evolutionary conserved mode of action. Altogether this suggests a role of TbEB1 in microtubule polymerization and indicates its putative involvement in cytokinesis

TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the flagellar pocket collar (FPC). TbBILBO1 is the first-described FPC protein of Trypanosoma brucei. It is essential for parasite survival, FP and FPC biogenesis. In this work, we characterize TbKINX1B, a novel TbBILBO1 partner. We demonstrate that TbKINX1B is located on the basal bodies, the microtubule quartet (a set of four microtubules) and the FPC in T. brucei. Down-regulation of TbKINX1B by RNA interference in bloodstream forms is lethal, inducing an overall disturbance in the endomembrane network. In procyclic forms, the RNAi knockdown of TbKINX1B leads to a minor phenotype with a small number of cells displaying epimastigote-like morphologies, with a misplaced kinetoplast. Our results characterize TbKINX1B as the first putative kinesin to be localized both at the basal bodies and the FPC with a potential role in transporting cargo along with the microtubule quartet