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

2017 ◽  
Vol 28 (13) ◽  
pp. 1782-1791 ◽  
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.

scholarly journals Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

2008 ◽  
Vol 181 (3) ◽  
pp. 431-438 ◽  
Author(s):  
Christopher L. de Graffenried ◽  
Helen H. Ho ◽  
Graham Warren
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Controlled Assembly

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.

Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei

1988 ◽  
Vol 90 (4) ◽  
pp. 577-589 ◽  
Author(s):  
R. Sasse ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Mitotic Spindle ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Alpha Tubulin ◽  
Daughter Cells ◽  
A Cell ◽  
Microtubular Organelles ◽  
Tubulin Content

We have used specific monoclonal antibodies to facilitate a study of acetylated and tyrosinated alpha-tubulin in the microtubule (MT) arrays in the Trypanosoma brucei cell. Acetylated alpha-tubulin is not solely located in the stable microtubular arrays but is present even in the ephemeral microtubules of the mitotic spindle. Moreover, there is a uniform distribution of this isoform in all arrays. Studies of flagella complexes show that acetylation is concomitant with assembly of MTs. There is no subsequent major modulation in the content of acetylated alpha-tubulin in MTs. Conversely, polymerizing flagellar MTs have a high tyrosinated alpha-tubulin content, which is subsequently reduced to a basal level at a discrete point in the cell cycle. The MTs of the intranuclear mitotic spindle appear never to contain tyrosinated alpha-tubulin, suggesting that they are actually constructed of detyrosinated alpha-tubulin or that detyrosination is extremely rapid at this time in the cell cycle. T. brucei therefore, represents a cell type with extremely active mechanisms for the post-translational modification of alpha-tubulin. Our analyses of the timing of acquisition and modulation in relation to MT construction in T. brucei, suggest that acetylation and detyrosination of alpha-tubulin are two independently regulated post-translational modifications, that are not uniquely associated with particular subsets of MTs of defined lability, position or function. Post-assembly detyrosination of alpha-tubulin may provide a mechanism whereby the cell could discriminate between new and old MTs, during construction of the cytoskeleton through the cell cycle. However, we also suggest that continuation of detyrosination, allows the cell, at cell division, to partition into daughter cells two equivalent sets of cytoskeletal MTs.

Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei

2001 ◽  
Vol 360 (1) ◽  
pp. 173-177 ◽  
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.

scholarly journals Golgi duplication in Trypanosoma brucei

2004 ◽  
Vol 165 (3) ◽  
pp. 313-321 ◽  
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.

scholarly journals Degradation of cyclin B is critical for nuclear division in Trypanosoma brucei

2017 ◽  
Author(s):  
Hanako Hayashi ◽  
Bungo Akiyoshi
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Nuclear Division ◽  
Spindle Checkpoint ◽  
Nuclear Genome ◽  
Single Copy ◽  
Cyclin B ◽  
Daughter Cells ◽  
Mitotic Cyclin ◽  
Assembly Defect

AbstractKinetoplastids have a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. These single-copy organelles must be duplicated and segregated faithfully to daughter cells at each cell division. In Trypanosoma brucei, although duplication of both organelles starts around the same time, segregation of the kinetoplast precedes that of the nucleus. Cytokinesis subsequently takes place so that daughter cells inherit a single copy of each organelle. Very little is known about the molecular mechanism that governs the timing of these events. Furthermore, it is thought that T. brucei lacks a spindle checkpoint that delays the onset of nuclear division in response to spindle damage. Here we show that a mitotic cyclin CYC6 has a dynamic localization pattern during the cell cycle, including kinetochore localization from G2 to metaphase. Using CYC6 as a molecular cell cycle marker, we confirmed that T. brucei cannot delay the onset of anaphase in response to a bipolar spindle assembly defect. Interestingly, expression of a stabilized form of CYC6 caused the nucleus to arrest in a metaphase-like state without preventing cytokinesis. We propose that trypanosomes have an ability to regulate the timing of nuclear division by modulating the CYC6 protein level, without a spindle checkpoint.

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
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.

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

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
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.

Bcl-2 protein localizes to the chromosomes of mitotic nuclei and is correlated with the cell cycle in cultured epithelial cell lines

1994 ◽  
Vol 107 (2) ◽  
pp. 363-371
Author(s):  
Q.L. Lu ◽  
A.M. Hanby ◽  
M.A. Nasser Hajibagheri ◽  
S.E. Gschmeissner ◽  
P.J. Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Epithelial Cell ◽  
Cell Lines ◽  
Cytoplasmic Localization ◽  
Human Epithelial Cell ◽  
Daughter Cells ◽  
Epithelial Cell Lines ◽  
Cell Immortalization

bcl-2 gene expression confers a survival advantage by preventing cells from entering apoptosis. In contrast to the previously described cytoplasmic localization of Bcl-2 in epithelial cells in vivo, in this study we have demonstrated, in a series of human epithelial cell lines, that Bcl-2 also localizes to mitotic nuclei. Both immunocytochemical and immunoelectron microscopical examinations localize this protein to nuclei and in particular to chromosomes. Nuclear Bcl-2 expression in these cell lines is correlated with the cell cycle. There is relatively strong expression during mitosis, most intense during prophase and metaphase, declining in telophase and then the protein becomes undetectable soon after separation of the two daughter cells. The expression and distribution of Bcl-2 is influenced by treatment with excessive thymidine. These results indicate that Bcl-2 may protect the cells from apoptosis occurring during mitosis and suggest a possible role for the protein in cell immortalization.

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

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.

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