scholarly journals Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in Trypanosoma brucei

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Xiaoduo Dong ◽  
Teck Kwang Lim ◽  
Qingsong Lin ◽  
Cynthia Y. He
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Spatial Organization ◽  
Screening Method ◽  
Interaction Network ◽  
General Mechanism ◽  
Basal Bodies ◽  
Body Protein ◽  
Content Type ◽  
Molecular Description

ABSTRACT Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In Trypanosoma brucei, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell cycle. Here, we investigated the interaction network of TbSpef1 using BioID, a proximity-dependent protein-protein interaction screening method. Characterization of selected candidates provided a molecular description of TbSpef1-MtQ interactions with nearby cytoskeletal structures. Of particular interest, we identified a new basal body protein TbSAF1, which is required for TbSpef1-MtQ anchorage to the basal bodies. The results demonstrate that MtQ-basal body anchorage is critical for the spatial organization of cytoskeletal organelles, as well as the morphology of the membrane-bound flagellar pocket where endocytosis takes place in this parasite. IMPORTANCE Trypanosoma brucei contains a large array of single-copied organelles and structures. Through extensive interorganelle connections, these structures replicate and divide following a strict temporal and spatial order. A microtubule quartet (MtQ) originates from the basal bodies and extends toward the anterior end of the cell, stringing several cytoskeletal structures together along its path. In this study, we examined the interaction network of TbSpef1, the only protein specifically located to the MtQ. We identified an interaction between TbSpef1 and a basal body protein TbSAF1, which is required for MtQ anchorage to the basal bodies. This study thus provides the first molecular description of MtQ association with the basal bodies, since the discovery of this association ∼30 years ago. The results also reveal a general mechanism of the evolutionarily conserved Spef1/CLAMP, which achieves specific cellular functions via their conserved microtubule functions and their diverse molecular interaction networks.

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 Sfr1, a Tetrahymena thermophila Sfi1 Repeat Protein, Modulates the Production of Cortical Row Basal Bodies

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Westley Heydeck ◽  
Alexander J. Stemm-Wolf ◽  
Janin Knop ◽  
Christina C. Poh ◽  
Mark Winey
Keyword(s):  
Basal Body ◽  
Binding Proteins ◽  
Tetrahymena Thermophila ◽  
Binding Protein ◽  
Extensive Study ◽  
Basal Bodies ◽  
Content Type ◽  
Inhibitory Role ◽  
Body Production

ABSTRACT Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood. Despite the extensive study of centrins in Tetrahymena thermophila, little is known about how centrin-binding proteins contribute to centrin’s roles in basal body assembly, stability, and orientation. The sole previous study of the large centrin-binding protein family in Tetrahymena revealed a role for Sfr13 in the stabilization and separation of basal bodies. In this study, we found that Sfr1 localizes to all Tetrahymena basal bodies and complete genetic deletion of SFR1 leads to overproduction of basal bodies. The uncovered inhibitory role of Sfr1 in basal body production suggests that centrin-binding proteins, as well as centrins, may influence basal body number both positively and negatively. Basal bodies are essential microtubule-based structures that template, anchor, and orient cilia at the cell surface. Cilia act primarily in the generation of directional fluid flow and sensory reception, both of which are utilized for a broad spectrum of cellular processes. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies of the ciliate Tetrahymena thermophila revealed important roles for two centrin family members in basal body assembly, separation of new basal bodies, and stability. Here, we characterize the basal body function of a centrin-binding protein, Sfr1, in Tetrahymena. Sfr1 is part of a large family of 13 proteins in Tetrahymena that contain Sfi1 repeats (SFRs), a motif originally identified in Saccharomyces cerevisiae Sfi1 that binds centrin. Sfr1 is the only SFR protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. In addition, Sfr1 resides predominantly at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Complete genomic knockout of SFR1 (sfr1Δ) causes a significant increase in both cortical row basal body density and the number of cortical rows, contributing to an overall overproduction of basal bodies. Reintroduction of Sfr1 into sfr1Δ mutant cells leads to a marked reduction of cortical row basal body density and the total number of cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study reveals an inhibitory role for Sfr1, and potentially centrins, in Tetrahymena basal body production. IMPORTANCE Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood. Despite the extensive study of centrins in Tetrahymena thermophila, little is known about how centrin-binding proteins contribute to centrin’s roles in basal body assembly, stability, and orientation. The sole previous study of the large centrin-binding protein family in Tetrahymena revealed a role for Sfr13 in the stabilization and separation of basal bodies. In this study, we found that Sfr1 localizes to all Tetrahymena basal bodies and complete genetic deletion of SFR1 leads to overproduction of basal bodies. The uncovered inhibitory role of Sfr1 in basal body production suggests that centrin-binding proteins, as well as centrins, may influence basal body number both positively and negatively.

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 Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Hung Quang Dang ◽  
Qing Zhou ◽  
Veronica W. Rowlett ◽  
Huiqing Hu ◽  
Kyu Joon Lee ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Functional Characterization ◽  
Body Protein ◽  
Superresolution Microscopy ◽  
Novel Proteins ◽  
Organelle Segregation ◽  
Body Components ◽  
Flagellar Axoneme

ABSTRACT The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei . Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote. IMPORTANCE The basal body in the early-branching protozoan Trypanosoma brucei nucleates flagellum assembly and also regulates organelle segregation, cell morphogenesis, and cell division. However, the molecular composition and the assembly process of the basal body remain poorly understood. Here, we identify 14 conserved basal body proteins and 25 trypanosome-specific basal body proteins via bioinformatics, localization-based screening, and proximity-dependent biotin identification. We further localized these proteins to distinct subdomains of the basal body by using fluorescence microscopy and superresolution microscopy, discovered novel regulators of basal body duplication and separation, and uncovered new functions of conserved basal body proteins in basal body duplication and separation. This work lays the foundation for dissecting the mechanisms underlying basal body biogenesis and inheritance in T. brucei .

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 Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

2010 ◽  
Vol 9 (6) ◽  
pp. 860-865 ◽  
Author(s):  
Lillian K. Fritz-Laylin ◽  
Zoe June Assaf ◽  
Sean Chen ◽  
W. Zacheus Cande
Keyword(s):  
Basal Body ◽  
De Novo ◽  
Unicellular Eukaryote ◽  
Basal Bodies ◽  
Microtubule Cytoskeleton ◽  
Cytoplasmic Microtubule ◽  
Content Type ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Naegleria Gruberi

ABSTRACT Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.

scholarly journals Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249908
Author(s):  
Catherine Sullenberger ◽  
Benjamin Hoffman ◽  
Justin Wiedeman ◽  
Gaurav Kumar ◽  
Kojo Mensa-Wilmot
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Basal Bodies ◽  
Kinetoplast Dna ◽  
Body Movements ◽  
Mitochondrial Nucleoid ◽  
Daughter Cells ◽  
Close Proximity ◽  
Division Factor ◽  
Mitochondrial Nucleoids

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

scholarly journals Analysis of a Mad2 homolog in Trypanosoma brucei provides possible hints on the origin of the spindle checkpoint

2020 ◽  
Author(s):  
Bungo Akiyoshi
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Nuclear Dna ◽  
Spindle Checkpoint ◽  
Nuclear Genome ◽  
26S Proteasome ◽  
Basal Bodies ◽  
Body Area ◽  
Uncharacterized Protein ◽  
Starting Point

AbstractThe spindle checkpoint is a surveillance mechanism that ensures accurate nuclear DNA segregation in eukaryotes. It does so by delaying the onset of anaphase until all kinetochores have established proper attachments to spindle microtubules. The evolutionary origin of the spindle checkpoint remains unclear. The flagellated kinetoplastid parasite Trypanosoma brucei has a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. The kinetoplast is physically linked to the basal body of the flagellum and its segregation is driven by the movement of basal bodies. While there is no strong evidence that T. brucei possesses a functional spindle checkpoint, it has been suggested that initiation of cytokinesis may be linked to the completion of kinetoplast segregation or basal body separation in this organism. Interestingly, the only identifiable spindle checkpoint component TbMad2 localizes at the basal body area. Here, I report identification of proteins that co-purified with TbMad2. One protein, which I propose to call TbMBP65, localizes at the basal body area and has a putative Mad2-interacting motif. Interestingly, 26S proteasome subunits also co-purified with TbMad2, raising a possibility that TbMad2 might regulate proteasome activity to regulate or monitor the segregation of basal bodies. I speculate that such a function might represent a prototype of the spindle checkpoint. I also show that TbAUK3, one of the three Aurora kinase homologs in T. brucei, localizes at the basal body area from late G1 until the time of kinetoplast separation. Immunoprecipitation of TbAUK3 identified an uncharacterized protein (termed TbABP79) that has a similar localization pattern as TbAUK3. These findings provide a starting point to reveal the function of TbMad2 and TbAUK3 as well as the origin of the spindle checkpoint.

Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

1992 ◽  
Vol 50 (1) ◽  
pp. 570-571
Author(s):  
Robert Hard ◽  
Gerald Rupp ◽  
Matthew L. Withiam-Leitch ◽  
Lisa Cardamone
Keyword(s):  
Basal Body ◽  
Untreated Control ◽  
Beat Frequency ◽  
Primary Cultures ◽  
Living Cells ◽  
Power Stroke ◽  
Ultrastructural Changes ◽  
Lung Cells ◽  
Basal Bodies ◽  
Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

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