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.

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 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 Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle.

1995 ◽  
Vol 128 (6) ◽  
pp. 1163-1172 ◽  
Author(s):  
D R Robinson ◽  
T Sherwin ◽  
A Ploubidou ◽  
E H Byard ◽  
K Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Daughter Nucleus ◽  
Basal Bodies ◽  
Cortical Microtubules ◽  
Cell Extension ◽  
Microtubule Polarity ◽  
Check Points ◽  
High Degree ◽  
Attachment Zone

Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.

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.

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 .

Does the oral apparatus of the ciliate Stentor inhibit oral development by release of a diffusible substance?

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 241-247
Author(s):  
Nöel De Terra
Keyword(s):  
Cell Division ◽  
Cell Surface ◽  
Basal Body ◽  
Basal Bodies ◽  
Glass Needle ◽  
Oral Development

In the ciliate Stentor, many thousands of basal bodies assemble on the ventral cell surface to form a new oral apparatus during cell division, regeneration and reorganization (oral replacement during interphase). During interphase, oral development is normally inhibited by the presence of the anteriorly placed oral apparatus. A glass needle was used to cut the oral apparatus of interphase stentors in two so that the parts remained intact but separate at the anterior end of the cell. These cells initiated basal body assembly and oral development, usually within 8 h. Basal body assembly can therefore result from disconnection of fibrous structures within the oral apparatus but is unlikely to be regulated by an inhibitor diffusing from it.

scholarly journals Katanin Knockdown Supports a Role for Microtubule Severing in Release of Basal Bodies before Mitosis in Chlamydomonas

2009 ◽  
Vol 20 (1) ◽  
pp. 379-388 ◽  
Author(s):  
M. Qasim Rasi ◽  
Jeremy D.K. Parker ◽  
Jessica L. Feldman ◽  
Wallace F. Marshall ◽  
Lynne M. Quarmby
Keyword(s):  
Cell Cycle ◽  
Rna Interference ◽  
Transition Zone ◽  
Basal Body ◽  
Cell Cycle Progression ◽  
Basal Bodies ◽  
Cycle Progression ◽  
Microtubule Severing ◽  
Regulation Of Cell Cycle ◽  
Precise Role

Katanin is a microtubule-severing protein that participates in the regulation of cell cycle progression and in ciliary disassembly, but its precise role is not known for either activity. Our data suggest that in Chlamydomonas, katanin severs doublet microtubules at the proximal end of the flagellar transition zone, allowing disengagement of the basal body from the flagellum before mitosis. Using an RNA interference approach we have discovered that severe knockdown of the p60 subunit of katanin, KAT1, is achieved only in cells that also carry secondary mutations that disrupt ciliogenesis. Importantly, we observed that cells in the process of cell cycle-induced flagellar resorption sever the flagella from the basal bodies before resorption is complete, and we find that this process is defective in KAT1 knockdown cells.

Cytoskeletal discontinuities in the cell body cortex initiate basal body assembly and oral development in the ciliate Stentor

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 249-257
Author(s):  
Nöel De Terra
Keyword(s):  
Cell Division ◽  
Cell Surface ◽  
Cell Body ◽  
Basal Body ◽  
Basal Bodies ◽  
Mass Assembly ◽  
Oral Development ◽  
Cortical Cytoskeleton

My previous work has shown that disconnecting the oral apparatus of Stentor into two parts induces mass assembly of basal bodies on the ventral cell surface and thus initiates oral development. This operation severs the extensive microtubule tracts joining the oral membranelles at their bases. To determine whether basal body assembly and oral development are also induced by permanently disconnecting the longitudinal microtubule fibre tracts (mt fibre tracts) of the cell body cortex, I interposed a ring of inverted (heteropolar) cortex between the anterior and posterior halves of interphase stentors. When successful, this operation made it impossible for these fibre tracts to rejoin at the heteropolar boundaries and always induced basal body assembly and oral development in the graft complex. By contrast, tripartite homopolar graft complexes rarely initiated oral development; when they did, it was apparently in response to the presence of disproportionately small oral structures, which is the normal stimulus for oral development in Stentor. The mt fibre tracts of tripartite homopolar grafts also eventually became continuous. These results support the hypothesis that permanent, extensive discontinuities anywhere within the cortical cytoskeleton can trigger basal body assembly and oral development. Since the onset of these processes is known to initiate cell division in Stentor, the results also suggest that development of discontinuities within the cortical cytoskeleton during interphase growth may be the endogenous stimulus initiating cell division in Stentor.

scholarly journals Tracking the Biogenesis and Inheritance of Subpellicular Microtubule inTrypanosoma bruceiwith Inducible YFP-α-Tubulin

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Omar Sheriff ◽  
Li-Fern Lim ◽  
Cynthia Y. He
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Trypanosoma Brucei ◽  
Daughter Cell ◽  
Structural System ◽  
Microtubule Cytoskeleton ◽  
Asymmetric Pattern ◽  
Microtubule Formation ◽  
Attachment Zone ◽  
Mode Of Inheritance

The microtubule cytoskeleton forms the most prominent structural system inTrypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance duringT. bruceicell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

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