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.

Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1

1999 ◽  
Vol 112 (21) ◽  
pp. 3769-3777 ◽  
Author(s):  
P. Bastin ◽  
T.J. Pullen ◽  
T. Sherwin ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Trypanosoma Brucei ◽  
Daughter Cell ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Transport Systems ◽  
Major Constituent ◽  
Wild Type ◽  
Detergent Extraction

The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or ‘blob’. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.

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 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 A grow-and-lock model for the control of flagellum length in trypanosomes

2018 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Benjamin Morga ◽  
Thierry Blisnick ◽  
Brice Rotureau ◽  
Philippe Bastin
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Daughter Cell ◽  
Linear Growth ◽  
Intraflagellar Transport ◽  
Elongation Rate ◽  
Eukaryotic Cells ◽  
Subsequent Increase ◽  
Normal Length ◽  
Cilia And Flagella

SUMMARYSeveral models have been proposed to explain how eukaryotic cells control the length of their cilia and flagella. Here, we investigated this process in the protistTrypanosoma brucei, an excellent system for cells with stable cilia like photoreceptors or spermatozoa. We show that the total amount of intraflagellar transport material (IFT, the machinery responsible for flagellum construction) increases during flagellum elongation, consistent with constant delivery of precursors and the previously reported linear growth. Reducing the IFT frequency by RNAi knockdown of the IFT kinesin motors slows down the elongation rate and results in the assembly of shorter flagella. These keep on elongating after cell division but fail to reach the normal length. This failure is neither due to an absence of precursors nor to a morphogenetic control by the cell body. We propose that the flagellum is locked after cell division, preventing further elongation or shortening. This is supported by the fact that subsequent increase in the IFT rate does not lead to further elongation. The distal tip FLAM8 protein was identified as a marker for the locking event. It is initiated prior cell division, leading to an arrest of elongation in the daughter cell. Here, we propose a new model termed grow-and-lock where the flagellum elongates until a locking event takes place in a timely defined manner hence fixing length. Alteration in the growth rate and/or in the timing of the locking event would lead to the formation of flagella of different lengths.

scholarly journals Par6α Interacts with the Dynactin Subunit p150Glued and Is a Critical Regulator of Centrosomal Protein Recruitment

2010 ◽  
Vol 21 (19) ◽  
pp. 3376-3385 ◽  
Author(s):  
Andrew Kodani ◽  
Vinh Tonthat ◽  
Beibei Wu ◽  
Christine Sütterlin
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Protein Delivery ◽  
Protein Transport ◽  
Protein Composition ◽  
Microtubule Cytoskeleton ◽  
Centrosomal Protein ◽  
Cycle Dependent ◽  
Protein Recruitment

The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle–dependent recruitment, retention, and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6α in protein transport to the centrosome. We detected Par6α at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150Glued. Depletion of Par6α caused the mislocalization of p150Glued and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6α and cell division was blocked. We propose a model in which Par6α controls centrosome organization through its association with the dynactin subunit p150Glued.

scholarly journals Polo-Like Kinase Guides Cytokinesis in Trypanosoma brucei through an Indirect Means

2010 ◽  
Vol 9 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Zhi Li ◽  
Takashi Umeyama ◽  
Ziyin Li ◽  
Ching C. Wang
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Critical Role ◽  
S Phase ◽  
Content Type ◽  
Synchronized Cells ◽  
Chromosomal Passenger ◽  
Attachment Zone ◽  
Target Effects ◽  
Rule Out

ABSTRACT Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the trans-localization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G1 phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.

scholarly journals Overlap of cargo binding sites on myosin V coordinates the inheritance of diverse cargoes

2012 ◽  
Vol 198 (1) ◽  
pp. 69-85 ◽  
Author(s):  
P. Taylor Eves ◽  
Yui Jin ◽  
Matthew Brunner ◽  
Lois S. Weisman
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Binding Sites ◽  
Daughter Cell ◽  
Common Mechanism ◽  
Myosin V ◽  
Yeast Vacuole ◽  
Cargo Binding

During cell division, organelles are distributed to distinct locations at specific times. For the yeast vacuole, the myosin V motor, Myo2, and its vacuole-specific cargo adaptor, Vac17, regulate where the vacuole is deposited and the timing of vacuole movement. In this paper, we show that Mmr1 functions as a mitochondria-specific cargo adaptor early in the cell cycle and that Mmr1 binds Myo2 at the site that binds Vac17. We demonstrate that Vac17 and Mmr1 compete for binding at this site. Unexpectedly, this competition regulates the volume of vacuoles and mitochondria inherited by the daughter cell. Furthermore, eight of the nine known Myo2 cargo adaptors overlap at one of two sites. Vac17 and Mmr1 overlap at one site, whereas Ypt11 and Kar9 bind subsets of residues that also bind Ypt31/Ypt32, Sec4, and Inp2. These observations predict that competition for access to Myo2 may be a common mechanism to coordinate the inheritance of diverse cargoes.

scholarly journals Regulation of the Cell Division Cycle in Trypanosoma brucei

2012 ◽  
Vol 11 (10) ◽  
pp. 1180-1190 ◽  
Author(s):  
Ziyin Li
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Spindle Assembly ◽  
Spindle Motor ◽  
Replication Initiation ◽  
Cell Division Cycle ◽  
Regulatory Pathways ◽  
Evolutionarily Conserved

ABSTRACT The cell division cycle is tightly regulated by the activation and inactivation of a series of proteins that control the replication and segregation of organelles to the daughter cells. During the past decade, we have witnessed significant advances in our understanding of the cell cycle in Trypanosoma brucei and how the cycle is regulated by various regulatory proteins. However, many other regulators, especially those unique to trypanosomes, remain to be identified, and we are just beginning to delineate the signaling pathways that drive the transitions through different cell cycle stages, such as the G 1 /S transition, G 2 /M transition, and mitosis-cytokinesis transition. Trypanosomes appear to employ both evolutionarily conserved and trypanosome-specific molecules to regulate the various stages of its cell cycle, including DNA replication initiation, spindle assembly, chromosome segregation, and cytokinesis initiation and completion. Strikingly, trypanosomes lack some crucial regulators that are well conserved across evolution, such as Cdc6 and Cdt1, which are involved in DNA replication licensing, the spindle motor kinesin-5, which is required for spindle assembly, the central spindlin complex, which has been implicated in cytokinesis initiation, and the actomyosin contractile ring, which is located at the cleavage furrow. Conversely, trypanosomes possess certain regulators, such as cyclins, cyclin-dependent kinases, and mitotic centromere-associated kinesins, that are greatly expanded and likely play diverse cellular functions. Overall, trypanosomes apparently have integrated unique regulators into the evolutionarily conserved pathways to compensate for the absence of those conserved molecules and, additionally, have evolved certain cell cycle regulatory pathways that are either different from its human host or distinct between its own life cycle forms.

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