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.

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

1986 ◽  
Vol 6 (3) ◽  
pp. 323-333 ◽  
Author(s):  
Christopher Bird ◽  
Marion Callus ◽  
Lynne Trickett ◽  
Robin Thorpe
Keyword(s):  
Monoclonal Antibody ◽  
Fibroblast Cell ◽  
Cytoskeletal Proteins ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Polymorphonuclear Leucocytes ◽  
Microfilament System ◽  
Fibroblast Cell Lines

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.

scholarly journals Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

1997 ◽  
Vol 24 (2) ◽  
pp. 399-410 ◽  
Author(s):  
Tomoko Kubori ◽  
Mitsumasa Okumura ◽  
Nobuhiro Kobayashi ◽  
Dai Nakamura ◽  
Masahiro Iwakura ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Basal Body ◽  
Purification And Characterization ◽  
Body Complex

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 Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium.

1985 ◽  
Vol 161 (3) ◽  
pp. 836-849 ◽  
Author(s):  
S I Aizawa ◽  
G E Dean ◽  
C J Jones ◽  
R M Macnab ◽  
S Yamaguchi
Keyword(s):  
Salmonella Typhimurium ◽  
Basal Body ◽  
Purification And Characterization ◽  
Body Complex

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 Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei

2015 ◽  
Vol 26 (17) ◽  
pp. 3013-3029 ◽  
Author(s):  
Michael R. McAllaster ◽  
Kyojiro N. Ikeda ◽  
Ana Lozano-Núñez ◽  
Dorothea Anrather ◽  
Verena Unterwurzacher ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Cytoskeletal Proteins ◽  
Sub Saharan Africa ◽  
Molecular Networks ◽  
Cell Morphogenesis ◽  
Daughter Cells ◽  
Sub Saharan ◽  
Attachment Zone

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.

Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei

2001 ◽  
Vol 114 (1) ◽  
pp. 141-148 ◽  
Author(s):  
K. Ersfeld ◽  
K. Gull
Keyword(s):  
Trypanosoma Brucei ◽  
Genetic Manipulation ◽  
Model System ◽  
Cytoskeletal Proteins ◽  
Related Protein ◽  
Relative Ease ◽  
Attachment Zone

The eukaryotic flagellum represents one of the most complex macromolecular structures found in any organism and contains more than 250 proteins. Due to the relative ease of genetic manipulation the flagellum of Trypanosoma brucei has emerged as an accessible model system to study the morphogenesis and dynamics of this organelle. We have recently started to characterise the mechanisms by which components of the cytoskeletal fraction of the flagellum, such as the axoneme, the paraflagellar rod and the flagellar attachment zone, are targeted by proteins synthesised in the cytoplasm and assembled. Here, we present the identification of a novel actin-related protein as a component of the axoneme. We show that this protein shares the tripeptid motif histidine-leucine-alanine (HLA) with one of the major proteins of the paraflagellar rod, PFRA. Building on previous work from this lab which showed that a deletion comprising this motif abolished targeting of PFRA to the flagellum we demonstrate in this study that the deletion of the tripeptid motif is sufficient to achieve mistargeting both of the PFRA and the actin-related protein. We propose that this motif represents an essential part of a flagellar targeting machinery in trypanosomes and possibly in other flagellated organisms.

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.

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