Giant FAZ10 is required for flagellum attachment zone stabilization and furrow positioning in Trypanosoma brucei

Abstract

The 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.

In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

“…This association is particularly relevant for FAZ filament proteins that display a 305 punctuated pattern in fluorescence. Following this idea, the absence of sticks at proximal locations as 306 observed in the present study does not agree with immunofluorescence data in which most of the FAZ 307 filament proteins were found to be present at equivalent proximal locations (Kohl et of FAZ10 is weaker than that of FAZ1 at proximal locations (Moreira et al, 2017). Since FAZ10 is a 310 giant protein (0.5 MDa) it is likely to have a significant contribution in the structure observed in electron 311 microscopy.…”

BOH1 cooperates with Polo-like kinase to regulate flagellum inheritance and cytokinesis initiation in Trypanosoma brucei

“…2C,D). This complicated the potential role of BOH1 in regulating cytokinesis, as flagellum detachment is known to also inhibit cytokinesis in T. brucei (LaCount et al, 2002;Moreira et al, 2017;Zhou et al, 2015Zhou et al, , 2011. Nonetheless, our finding that BOH1 deficiency disrupted TbPLK localization to the FAZ tip (Fig.…”

“…The flagellum and its associated cytoskeletal structures, including the basal body, the FAZ, the flagellar pocket and the bilobe structure consisting of a hook complex and a centrin arm, are duplicated during the S phase of the cell cycle, and are segregated thereafter during G2 and mitotic phases. While the assembly of the new flagellum requires duplication of the basal body (Dang et al, 2017;Hu et al, 2015a), correct positioning and attachment of the newly assembled flagellum is impacted by the faithful duplication and segregation of other flagellum-associated cytoskeletal structures, such as the FAZ and the bilobe structure (Lacomble et al, 2012;Moreira et al, 2017;Rotureau et al, 2014;Sun et al, 2013;Vaughan et al, 2008;Zhou et al, 2010Zhou et al, , 2015Zhou et al, , 2011. This important cellular process is governed by reversible protein phosphorylation mediated by the Polo-like kinase homolog TbPLK (Ikeda and de Graffenried, 2012) and the putative protein phosphatase kinetoplastid-specific protein phosphatase 1 named KPP1 (Zhou et al, 2018b), both of which localize to the basal body, the hook complex and the distal tip of the new FAZ, and may regulate certain downstream factors at these cytoskeletal structures.…”

Functional analyses of the CIF1-CIF2 complex in Trypanosoma brucei identify the structural motifs required for complex formation and cytokinesis

“…The molecular mechanism for the unidirectional, longitudinal binary cell fission in trypanosome remains poorly understood. Based on the observed cell cycle defects through RNAimediated gene ablation, many proteins have been reported to be involved in cytokinesis, which localize to various subcellular structures and possess diverse biochemical functions (5,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). However, many of these proteins localize to the cytosol, raising the question of whether they play a direct role in cytokinesis.…”