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 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 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.

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 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 Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

2020 ◽  
Author(s):  
Clare Halliday ◽  
Ryuji Yanase ◽  
Carolina Moura Costa Catta-Preta ◽  
Flavia Moreira-Leite ◽  
Jitka Myskova ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Membrane ◽  
Cell Body ◽  
Cell Shape ◽  
Light And Electron Microscopy ◽  
Sand Fly ◽  
Ecological Niches ◽  
Flagellar Pocket ◽  
Cell Shapes ◽  
Attachment Zone

AbstractThe shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

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.

Evidence for a Mr 88,000 glycoprotein with a transmembrane association to a unique flagellum attachment region in Trypanosoma brucei

1989 ◽  
Vol 93 (3) ◽  
pp. 501-508
Author(s):  
A. Woods ◽  
A.J. Baines ◽  
K. Gull
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Body ◽  
Outer Surface ◽  
Fluorescein Isothiocyanate ◽  
Surface Glycoprotein ◽  
Narrow Line ◽  
Variable Surface ◽  
Attachment Region ◽  
Relationship Of ◽  
Attachment Zone

We have examined the relationship of externally accessible proteins associated with the internal cytoskeleton of procyclic Trypanosoma brucei. Two approaches were taken. First, externally disposed glycoproteins were identified with lectins and examined for their persistence and location in isolated cytoskeletons. Second, proteins containing tyrosine residues available for chemical modification on the outer surface were identified in isolated cytoskeletons and probed for glycosylation. The procyclic form of T. brucei that was employed does not express the variable surface glycoprotein. The lectin concanavalin A (ConA) bound to the outer surface of T. brucei in two discrete locations; one a narrow line close to the flagellum attachment zone on the cell body, the other at the distal tip of the flagellum itself. Of these, only the cell body labelling was detected when isolated cytoskeletons were probed with fluorescein isothiocyanate-labelled ConA. When cytoskeletons were prepared from cells labelled with gold-conjugated ConA, a narrow line of label was detected parallel to the flagellum attachment zone but was distinct from it. Only one cytoskeletal protein, of Mr 88,000, could be labelled at the cell surface by the 125I/iodogen procedure. This protein could be precipitated from SDS-solubilized cytoskeletons with ConA-agarose. These data indicate the existence of a previously undetected cytoskeletal structure, situated in the cell body, close to the point of flagellum attachment, which has a transmembrane association with an external Mr 88,000 glycoprotein.

scholarly journals In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

2020 ◽  
Author(s):  
Sylvain Trépout
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Trypanosoma Brucei ◽  
Cell Body ◽  
Electron Tomography ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Attachment Zone

SummaryThe flagellum of Trypanosoma brucei is a 20 µm-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at high resolution in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights in the structure the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.HighlightsFlagellar and cellular membranes are in close contact next to the FAZ filamentSticks are heterogeneously distributed along the FAZ filament lengthThin appendages are present between the FAZ filament sticks to neighbouring microtubulesFAZ elongation could originate from the force exerted by dynein motors on subpellicular microtubules

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.

Sign in / Sign up

Export Citation Format

Share Document