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The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubulebinding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis. [ABSTRACT FROM AUTHOR]

The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell-cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQassociated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQassociated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei. [ABSTRACT FROM AUTHOR]

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 trypanosomespecific 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. [ABSTRACT FROM AUTHOR]

To proliferate, the parasitic protozoan Trypanosoma brucei undergoes binary fission in a unidirectional manner along the cell's longitudinal axis from the cell anterior toward the cell posterior. This unusual mode of cell division is controlled by a regulatory pathway composed of two evolutionarily conserved protein kinases, Polo-like kinase and Aurora B kinase, and three trypanosome-specific proteins, CIF1, CIF2, and CIF3, which act in concert at the cytokinesis initiation site located at the distal tip of the newly assembled flagellum attachment zone (FAZ). However, additional regulators that function in this cytokinesis signaling cascade remain to be identified and characterized. Using proximity biotinylation, co-immunofluorescence microscopy, and co-immunoprecipitation, we identified 52 CIF1-associated proteins and validated six CIF1-interacting proteins, including the putative protein phosphatase KPP1, the katanin p80 subunit KAT80, the cleavage furrow--localized proteins KLIF and FRW1, and the FAZ tip--localized proteins FAZ20 and FPRC. Further analyses of the functional interplay between CIF1 and its associated proteins revealed a requirement of CIF1 for localization of a set of CIF1-associated proteins, an interdependence between KPP1 and CIF1, and an essential role of katanin in the completion of cleavage furrow ingression. Together, these results suggest that CIF1 acts as a master regulator of cytokinesis in T. brucei by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site. [ABSTRACT FROM AUTHOR]

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Pham et al.)

To proliferate, the parasitic protozoan Trypanosoma brucei undergoes binary fission in a unidirectional manner along the cell's longitudinal axis from the cell anterior toward the cell posterior. This unusual mode of cell division is controlled by a regulatory pathway composed of two evolutionarily conserved protein kinases, Polo-like kinase and Aurora B kinase, and three trypanosome-specific proteins, CIF1, CIF2, and CIF3, which act in concert at the cytokinesis initiation site located at the distal tip of the newly assembled flagellum attachment zone (FAZ). However, additional regulators that function in this cytokinesis signaling cascade remain to be identified and characterized. Using proximity biotinylation, co-immunofluorescence microscopy, and co-immunoprecipitation, we identified 52 CIF1-associated proteins and validated six CIF1-interacting proteins, including the putative protein phosphatase KPP1, the katanin p80 subunit KAT80, the cleavage furrow-localized proteins KLIF and FRW1, and the FAZ tip-localized proteins FAZ20 and FPRC. Further analyses of the functional interplay between CIF1 and its associated proteins revealed a requirement of CIF1 for localization of a set of CIF1-associated proteins, an interdependence between KPP1 and CIF1, and an essential role of katanin in the completion of cleavage furrow ingression. Together, these results suggest that CIF1 acts as a master regulator of cytokinesis in T. brucei by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site., (© 2018 Zhou et al.)