scholarly journals KMP-11, a Basal Body and Flagellar Protein, Is Required for Cell Division in Trypanosoma brucei

2008 ◽  
Vol 7 (11) ◽  
pp. 1941-1950 ◽  
Author(s):  
Ziyin Li ◽  
Ching C. Wang
Keyword(s):  
Cell Division ◽  
Rna Interference ◽  
Membrane Protein ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Nuclear Division ◽  
Procyclic Form ◽  
Similar Phenotype ◽  
Flagellar Protein ◽  
Attachment Zone

ABSTRACT Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.

scholarly journals An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in Trypanosoma brucei

2013 ◽  
Vol 24 (9) ◽  
pp. 1321-1333 ◽  
Author(s):  
Ana Lozano-Núñez ◽  
Kyojiro N. Ikeda ◽  
Thomas Sauer ◽  
Christopher L. de Graffenried
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Rna Interference ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Basal Bodies ◽  
Body Rotation ◽  
The Many ◽  
Attachment Zone

Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLKas) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.

scholarly journals Trypanosomes have divergent kinesin-2 proteins that function differentially in IFT, cell division, and motility

2018 ◽  
Author(s):  
Robert L. Douglas ◽  
Brett M. Haltiwanger ◽  
Haiming Wu ◽  
Robert L. Jeng ◽  
Joel Mancuso ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Division ◽  
Trypanosoma Brucei ◽  
Cell Body ◽  
Basal Body ◽  
Protein Function ◽  
Intraflagellar Transport ◽  
Tail Domain ◽  
Specialized Structure ◽  
Attachment Zone

SummaryTrypanosoma brucei, the causative agent of African sleeping sickness, has a flagellum that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 motor proteins. In this study, we investigated the function of the two T. brucei kinesin-2 proteins, TbKin2a and TbKin2b, in bloodstream form trypanosomes. We found that compared to other kinesin-2 proteins, TbKin2a and TbKin2b show greater variation in neck, stalk, and tail domain sequences. Both kinesins contributed additively to flagellar lengthening. Surprisingly, silencing TbKin2a inhibited cell proliferation, cytokinesis and motility, whereas silencing TbKin2b did not. TbKin2a was localized on the flagellum and colocalized with IFT components near the basal body, consistent with it performing a role in IFT. TbKin2a was also detected on the flagellar attachment zone, a specialized structure in trypanosome cells that connects the flagellum to the cell body. Our results indicate that kinesin-2 proteins in trypanosomes play conserved roles in IFT and exhibit a specialized localization, emphasizing the evolutionary flexibility of motor protein function in an organism with a large complement of kinesins.

Immunological characterization of cytoskeletal proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 77-85 ◽  
Author(s):  
R. Woodward ◽  
M. J. Carden ◽  
K. Gull
Keyword(s):  
Monoclonal Antibody ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Complex Mixture ◽  
Cytoskeletal Proteins ◽  
Bovine Sperm ◽  
Phosphatase Treatment ◽  
Body Complex ◽  
Attachment Zone

SUMMARYThe monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

The cell division cycle of Trypanosoma brucei brucei : timing of event markers and cytoskeletal modulations

1989 ◽  
Vol 323 (1218) ◽  
pp. 573-588 ◽  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Single Copy ◽  
Cell Division Cycle ◽  
Basal Bodies ◽  
Division Plane ◽  
Transmission Electron ◽  
Single Nucleus

We have analysed the timing and order of events occurring within the cell division cycle of Trypanosoma brucei . Cells in the earliest stages of the cell cycle possess a single copy of three major organelles: the nucleus, the kinetoplast and the flagellum. The first indication of progress through the cell cycle is the elongation of the pro-basal body lying adjacent to the mature basal body subtending the flagellum. This newly elongated basal body occupies a posterior position within the cell when it initiates growth of the new daughter flagellum. Genesis of two new pro-basal bodies occurs only after growth of the new daughter flagellum has been initiated. Extension of the new flagellum, together with the paraflagellar rod, then continues throughout a major portion of the cell cycle. During this period of flagellum elongation, kinetoplast division occurs and the two kinetoplasts, together with the two flagellar basal bodies, then move apart within the cell. Mitosis is then initiated and a complex pattern of organelle positions is achieved whereby a division plane runs longitudinally through the cell such that each daughter ultimately receives a single nucleus, kinetoplast and flagellum. These events have been described from observations of whole cytoskeletons by transmission electron microscopy together with detection of particular organelles by fluorescence microscopy. The order and timing of events within the cell cycle has been derived from analyses of the proportion of a given cell type occurring within an exponentially growing culture.

scholarly journals Polo-Like Kinase Is Expressed in S/G2/M Phase and Associated with the Flagellum Attachment Zone in both Procyclic and Bloodstream Forms of Trypanosoma brucei

2008 ◽  
Vol 7 (9) ◽  
pp. 1582-1590 ◽  
Author(s):  
Takashi Umeyama ◽  
Ching C. Wang
Keyword(s):  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Initiation Site ◽  
Etiologic Agent ◽  
Enhanced Yellow Fluorescent Protein ◽  
Procyclic Form ◽  
Distinct Cell ◽  
M Phase ◽  
Attachment Zone

ABSTRACTTrypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue inT.brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G2/M phase, and vanishes in G1phase. An indicated TbPLK-EYFP association with the FAZs of G2/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.

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

2012 ◽  
Vol 12 (2) ◽  
pp. 168-181 ◽  
Author(s):  
Anaïs Brasseur ◽  
Brice Rotureau ◽  
Marjorie Vermeersch ◽  
Thierry Blisnick ◽  
Didier Salmon ◽  
...  
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Normal Cell ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Flagellar Pocket ◽  
Content Type ◽  
Procyclic Form ◽  
Specific Localization ◽  
Inside Out

ABSTRACT 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.

scholarly journals The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

2015 ◽  
Vol 14 (9) ◽  
pp. 898-907 ◽  
Author(s):  
Huiqing Hu ◽  
Yi Liu ◽  
Qing Zhou ◽  
Sara Siegel ◽  
Ziyin Li
Keyword(s):  
Cell Cycle ◽  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Essential Role ◽  
Microtubule Organizing Center ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Organizing Center

ABSTRACT The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei , an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei .

scholarly journals Centrin1 Is Required for Organelle Segregation and Cytokinesis in Trypanosoma brucei

2007 ◽  
Vol 18 (9) ◽  
pp. 3290-3301 ◽  
Author(s):  
Angamuthu Selvapandiyan ◽  
Praveen Kumar ◽  
James C. Morris ◽  
Jeffrey L. Salisbury ◽  
Ching C. Wang ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Calcium Binding ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Procyclic Form ◽  
Organelle Segregation ◽  
Leishmania Amastigotes ◽  
First Time

Centrin is a calcium-binding centrosome/basal body–associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.

scholarly journals Clathrin-Dependent Targeting of Receptors to the Flagellar Pocket of Procyclic-Form Trypanosoma brucei

2004 ◽  
Vol 3 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Chien-Hui Hung ◽  
Xugang Qiao ◽  
Pei-Tseng Lee ◽  
Mary Gwo-Shu Lee
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Secretory Pathway ◽  
Morphological Changes ◽  
Cell Receptor ◽  
Flagellar Pocket ◽  
Procyclic Form ◽  
Receptor Mediated Endocytosis ◽  
Distinct Structure ◽  
Endocytic Vesicles

ABSTRACT In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.

scholarly journals Cell Cycle-dependent Nuclear Localization of Yeast RNase III Is Required for Efficient Cell Division

2004 ◽  
Vol 15 (7) ◽  
pp. 3015-3030 ◽  
Author(s):  
Mathieu Catala ◽  
Bruno Lamontagne ◽  
Stéphanie Larose ◽  
Ghada Ghazal ◽  
Sherif Abou Elela
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Rna Interference ◽  
Cell Cycle Progression ◽  
Nuclear Division ◽  
Single Mutation ◽  
Rnase Iii ◽  
Independent Functions ◽  
M Phase ◽  
Cycle Dependent

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Δrnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.

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