Abstract
ABSTRACTThe flagellum and flagellum attachment zone (FAZ) are important cytoskeletal structures in trypanosomatids, being required for motility, cell division and cell morphogenesis. Trypanosomatid cytoskeletons contain abundant high molecular mass proteins (HMMPs), but many of their biological functions are still unclear. Here, we report the characterization of the giant FAZ protein, FAZ10, in Trypanosoma brucei, which, using immunoelectron microscopy, we show localizes to the intermembrane staples in the FAZ intracellular domain. Our data show that FAZ10 is a giant cytoskeletal protein essential for normal growth and morphology in both procyclic and bloodstream parasite life cycle stages, with its depletion leading to defects in cell morphogenesis, flagellum attachment, and kinetoplast and nucleus positioning. We show that the flagellum attachment defects are probably brought about by reduced tethering of the proximal domain of the paraflagellar rod to the FAZ filament. Further, FAZ10 depletion also reduces abundance of FAZ flagellum domain protein, ClpGM6. Moreover, ablation of FAZ10 impaired the timing and placement of the cleavage furrow during cytokinesis, resulting in premature or asymmetrical cell division.
Highlights
Trypanosoma brucei is the causative agent of human African trypanosomiasis and belongs to the Trypanosomatidae family
Trypanosoma brucei FAZ10 is a large and repetitive cytoskeletal protein A mass spectrometry analysis of T. brucei high molecular mass proteins (HMMPs) (>500 kDa) excised from an SDS-PAGE gel was performed and the resulting peptide data was matched against the TriTrypDB (Aslett et al, 2010)
FAZ10 is encoded by a 13,005 bp long ORF on chromosome 7 and is predicted to be a HMMP (≥502 kDa) based on its sequence in TriTrypDB
Summary
Trypanosoma brucei is the causative agent of human African trypanosomiasis and belongs to the Trypanosomatidae family. The cytoskeleton is a well-defined component of the cell with two main structures: a subpellicular corset of microtubules that defines cell shape and size, and a flagellum that is crucial for motility and cell division (Hemphill et al, 1991). The cytoskeleton undergoes rearrangement during cell morphogenesis and division, making this structure essential for trypanosome biology (Sherwin and Gull, 1989). It is known that it is a large and complex interconnected set of fibres, filaments and junctional complexes that link the flagellum to the cell body, and that comprises five main domains (FAZ flagellum domain, FAZ intracellular domain, FAZ filament domain, microtubule quartet domain and microtubule quartet-FAZ linker domain) that can be subdivided into eight overlapping zones (Sunter and Gull, 2016). It is important to identify and characterize new components of each of these zones in order to understand FAZ biogenesis and function
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