Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex
Abstract
:1. Introduction
2. Methods
2.1. Cell Lines, Culture and Transfection
2.2. Anti-BHALIN Antibody Production
2.3. Reporter Uptake Assays
2.4. Immunofluorescence Assay
2.5. Molecular Biology and Cloning
2.6. Cloning Full-Length BHALIN and Truncations for Transfection into U-2 OS Cells
2.7. Extraction of RNA
2.8. Semi-Quantitative RT-PCR
2.9. Western Blot Analysis
2.10. Yeast Two-Hybrid Assay
3. Results
3.1. BHALIN Is a Trypanosomatid Specific Protein
3.2. BHALIN Forms a Hook-Shaped Structure at the Hook Complex
3.3. BHALIN Is Present at the Hook Complex throughout the Cell Cycle
3.4. Binding of BHALIN Modifies the Shape of the TbBILBO1 Filaments and Requires the TbBILBO1 EF-Hand Domain
3.5. Knockdown of BHALIN in Procyclic Forms Leads to an Altered Cell Cycle Profile
3.6. Knockdown of BHALIN in Bloodstream Forms Is Lethal and Induces a “BigEye” Phenotype
3.7. Endocytosis Is Perturbed in BSF BHALIN RNAi Cells
3.8. Expression of RNAi-Resistant BHALIN Rescues the Lethal Phenotype in BSF
3.9. Deletion of the BILBO1-Binding and C-Terminal Domains Relocates BHALIN to the Cytoplasm
3.10. The BHALIN TbBILBO1-Binding Domain (T2) Co-Localises with TbBILBO1 In Vivo
3.11. Expression of BHALIN BILBO1-Binding Domain in BSF Does Not Rescue the RNAi-Induced Lethal Phenotype
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Broster Reix, C.E.; Florimond, C.; Cayrel, A.; Mailhé, A.; Agnero-Rigot, C.; Landrein, N.; Dacheux, D.; Havlicek, K.; Bonhivers, M.; Morriswood, B.; et al. Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex. Microorganisms 2021, 9, 2334. https://doi.org/10.3390/microorganisms9112334
Broster Reix CE, Florimond C, Cayrel A, Mailhé A, Agnero-Rigot C, Landrein N, Dacheux D, Havlicek K, Bonhivers M, Morriswood B, et al. Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex. Microorganisms. 2021; 9(11):2334. https://doi.org/10.3390/microorganisms9112334
Chicago/Turabian StyleBroster Reix, Christine E., Célia Florimond, Anne Cayrel, Amélie Mailhé, Corentin Agnero-Rigot, Nicolas Landrein, Denis Dacheux, Katharina Havlicek, Mélanie Bonhivers, Brooke Morriswood, and et al. 2021. "Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex" Microorganisms 9, no. 11: 2334. https://doi.org/10.3390/microorganisms9112334