scholarly journals CRL4WDR1 Controls Polo-like Kinase Protein Abundance to Promote Bilobe Duplication, Basal Body Segregation and Flagellum Attachment in Trypanosoma brucei

2017 ◽  
Vol 13 (1) ◽  
pp. e1006146 ◽  
Author(s):  
Huiqing Hu ◽  
Qing Zhou ◽  
Xianxian Han ◽  
Ziyin Li
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Protein Abundance

scholarly journals NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei

2006 ◽  
Vol 119 (9) ◽  
pp. 1852-1863 ◽  
Author(s):  
L. C. Pradel
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body

Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei

1990 ◽  
Vol 95 (1) ◽  
pp. 49-57 ◽  
Author(s):  
R. Woodward ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Unit Cell ◽  
Kinetoplast Dna ◽  
Interphase Cell ◽  
Body Formation ◽  
Timing Of Initiation ◽  
Single Mitochondrion

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei

1999 ◽  
Vol 112 (24) ◽  
pp. 4687-4694 ◽  
Author(s):  
V. Dilbeck ◽  
M. Berberof ◽  
A. Van Cauwenberge ◽  
H. Alexandre ◽  
E. Pays
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Coiled Coil ◽  
Western Blots ◽  
Cell Extracts ◽  
Acid Protein ◽  
Coil Structure ◽  
Tubulin Antibodies ◽  
Body Component ◽  
Gamma Tubulin

TBBC (for Trypanosoma brucei basal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are (gamma)-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-(gamma)-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.

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.

scholarly journals Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei

2019 ◽  
Vol 295 (3) ◽  
pp. 729-742
Author(s):  
Kieu T. M. Pham ◽  
Ziyin Li
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Super Resolution ◽  
Specific Protein ◽  
Protein Stabilization ◽  
Structured Illumination ◽  
Human Parasite ◽  
Evolutionarily Conserved ◽  
Body Components ◽  
Super Resolution Microscopy

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

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 Ultrastructure expansion microscopy in Trypanosoma brucei

Open Biology ◽  
2021 ◽  
Vol 11 (10) ◽  
Author(s):  
Ana Kalichava ◽  
Torsten Ochsenreiter
Keyword(s):  
Endoplasmic Reticulum ◽  
Sample Preparation ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Microscopic Imaging ◽  
Future Studies ◽  
Challenges And Opportunities ◽  
A Cell ◽  
Isotropic Expansion ◽  
Physical Expansion

The recently developed ultrastructure expansion microscopy (U-ExM) technique allows us to increase the spatial resolution within a cell or tissue for microscopic imaging through the physical expansion of the sample. In this study, we validate the use of U-ExM in Trypanosoma brucei measuring the expansion factors of several different compartments/organelles and thus verify the isotropic expansion of the cell. We furthermore demonstrate the use of this sample preparation protocol for future studies by visualizing the nucleus and kDNA, as well as proteins of the cytoskeleton, the basal body, the mitochondrion and the endoplasmic reticulum. Lastly, we discuss the challenges and opportunities of U-ExM.

scholarly journals An Evolutionarily Conserved Coiled-Coil Protein Implicated in Polycystic Kidney Disease Is Involved in Basal Body Duplication and Flagellar Biogenesis in Trypanosoma brucei

2005 ◽  
Vol 25 (9) ◽  
pp. 3774-3783 ◽  
Author(s):  
Gareth W. Morgan ◽  
Paul W. Denny ◽  
Sue Vaughan ◽  
David Goulding ◽  
Tim R. Jeffries ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Critical Role ◽  
Coiled Coil ◽  
Specific Protein ◽  
Polycystic Kidney ◽  
Wide Range ◽  
Flagellar Biogenesis

ABSTRACT Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.

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