Knockdown of APC/C-associated genes and its effect on viability and cell cycle of protozoan parasite of Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.

Download Full-text

A role for Sar1 and ARF1 GTPases during Golgi biogenesis in the protozoan parasite Trypanosoma brucei

Molecular Biology of the Cell ◽

10.1091/mbc.e17-03-0151 ◽

2017 ◽

Vol 28 (13) ◽

pp. 1782-1791 ◽

Cited By ~ 2

Author(s):

Sevil Yavuz ◽

Graham Warren

Keyword(s):

Cell Cycle ◽

Trypanosoma Brucei ◽

Protozoan Parasite ◽

Small Gtpases ◽

Intact Cells ◽

Golgi Stack ◽

Daughter Cells ◽

Photoactivatable Gfp

A single Golgi stack is duplicated and partitioned into two daughter cells during the cell cycle of the protozoan parasite Trypanosoma brucei. The source of components required to generate the new Golgi and the mechanism by which it forms are poorly understood. Using photoactivatable GFP, we show that the existing Golgi supplies components directly to the newly forming Golgi in both intact and semipermeabilized cells. The movement of a putative glycosyltransferase, GntB, requires the Sar1 and ARF1 GTPases in intact cells. In addition, we show that transfer of GntB from the existing Golgi to the new Golgi can be recapitulated in semipermeabilized cells and is sensitive to the GTP analogue GTPγS. We suggest that the existing Golgi is a key source of components required to form the new Golgi and that this process is regulated by small GTPases.

Download Full-text

Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei

Biochemical Journal ◽

10.1042/bj3600173 ◽

2001 ◽

Vol 360 (1) ◽

pp. 173-177 ◽

Cited By ~ 14

Author(s):

Mostafa KABIRI ◽

Dietmar STEVERDING

Keyword(s):

Cell Cycle ◽

Superoxide Dismutase ◽

Trypanosoma Brucei ◽

Protozoan Parasite ◽

Small Subunit ◽

Developmentally Regulated ◽

Iron Superoxide Dismutase ◽

Its Gene ◽

Quantitative Changes ◽

Post Transcriptional Regulation

An iron superoxide dismutase (FeSOD) gene of the protozoan parasite Trypanosoma brucei has been cloned and its gene product functionally characterized. The gene encodes a protein of 198 residues which shows 80% identity with FeSODs from other trypanosomatids. Inhibitor studies with purified recombinant FeSOD expressed in Escherichia coli confirmed that the enzyme is an iron-containing SOD. The FeSOD is developmentally regulated in the parasite, expression being lowest in the cell-cycle-arrested, short stumpy bloodstream forms. Differential expression of the FeSOD protein contrasts with only minor quantitative changes in the FeSOD mRNA, indicating post-transcriptional regulation of the enzyme. As the level of FeSOD increases during differentiation of cell-cycle-arrested short stumpy into dividing procyclic forms, it is suggested that the enzyme is only required in proliferating stages of the parasite for the elimination of superoxide radicals which are released during the generation of the iron-tyrosyl free-radical centre in the small subunit of ribonucleotide reductase.

Download Full-text

Golgi duplication in Trypanosoma brucei

The Journal of Cell Biology ◽

10.1083/jcb.200311076 ◽

2004 ◽

Vol 165 (3) ◽

pp. 313-321 ◽

Cited By ~ 109

Author(s):

Cynthia Y. He ◽

Helen H. Ho ◽

Joerg Malsam ◽

Cecile Chalouni ◽

Christopher M. West ◽

...

Keyword(s):

Cell Cycle ◽

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Trypanosoma Brucei ◽

Basal Body ◽

Time Course ◽

Matrix Protein ◽

De Novo ◽

Protozoan Parasite ◽

Er Export

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.

Download Full-text

Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽

10.12688/f1000research.10342.2 ◽

2017 ◽

Vol 6 ◽

pp. 683 ◽

Cited By ~ 31

Author(s):

Terry K. Smith ◽

Frédéric Bringaud ◽

Derek P. Nolan ◽

Luisa M. Figueiredo

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protozoan Parasite ◽

Tsetse Fly ◽

Metabolic Reprogramming ◽

Mammalian Host ◽

Insect Vector ◽

Environmental Signals ◽

Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

Download Full-text

Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8191 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8191-8200 ◽

Cited By ~ 63

Author(s):

Philippe Bastin ◽

Thomas H. MacRae ◽

Susan B. Francis ◽

Keith R. Matthews ◽

Keith Gull

Keyword(s):

Cell Cycle ◽

Trypanosoma Brucei ◽

Protein Targeting ◽

Fluorescent Protein ◽

Green Fluorescent Protein Reporter ◽

Mutant Proteins ◽

Green Fluorescent ◽

A Minor ◽

Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma 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.

Download Full-text

Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092912 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C708-C708

Author(s):

Cho Yeow Koh ◽

Jasmine Nguyen ◽

Sayaka Shibata ◽

Zhongsheng Zhang ◽

Ranae Ranade ◽

...

Keyword(s):

Crystal Structures ◽

Trypanosoma Brucei ◽

High Throughput ◽

High Throughput Screening ◽

Protozoan Parasite ◽

Trna Synthetase ◽

Structure Based Drug Design ◽

Chemical Structures ◽

Ic50 Values ◽

Methionyl Trna Synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

Download Full-text