A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation

Abstract

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 MtQ-associated 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 MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.

Keywords: Trypanosoma brucei; basal body; flagellum inheritance; microtubule quartet.

Figure 1

The organization, duplication, and segregation of flagellum and flagellum-associated cytoskeletal structures during T. brucei cell cycle.A, schematic drawing of the flagellum and flagellum-associated cytoskeletal structures in T. brucei. The scale bar represents 5 μm. B, schematic drawing of the duplication and segregation of flagellum and flagellum-associated structures during T. brucei cell cycle. NFD, new-flagellum daughter; OFD, old-flagellum daughter.

Figure 2

NHL1 localizes to the proximal portion of the microtubule quartet (MtQ).A, schematic drawing of the structural motif in NHL1 (upper panel) and homology modeling of the NHL-like domain in NHL1 (lower panel) using SWISS model. The template used for modeling was 6G7W. B, localization of NHL1 relative to various flagellum-associated cytoskeletal structures by immunofluorescence microscopy. Endogenous NHL1-3HA was detected by FITC-conjugated anti-HA antibody. Anti-TbCentrin4 labels label basal body (BB) and centrin arm (CA), anti-TbMORN1 labels hook complex (HC), and anti-TbSAS-6 labels mature basal body (mBB) and pro-basal body (pBB). Endogenous PTP-TbSpef1 was detected by anti–protein A antibody to label the proximal portion of the MtQ. The scale bar represents 5 μm. The scale bar in the zoom-in image represents 1 μm. C, 3D-SIM to examine the localization of NHL1 relative to the basal body and the centrin arm labeled by anti-TbCentrin4. Scale bar: 1 μm. D, 3D-SIM to detect the localization of NHL1 relative to TbSpef1. The scale bar represents 1 μm. HA, hemagglutinin.

Figure 3

NHL1 is required for completion of cytokinesis.A, RNAi of NHL1 in procyclic trypanosomes. Endogenous NHL1-3HA was detected by anti-HA antibody. TbPSA6 was detected by anti-TbPSA6 as a loading control. B, effect of NHL1 RNAi on cell proliferation. Shown is the growth curve of NHL1 RNAi cell line, a representative clone of two clonal RNAi cell lines, which showed similar growth defects. (n = 3). C, effect of NHL1 knockdown on cell cycle progression. Shown is the counting of cells with different numbers of nuclei (N) and kinetoplasts (K). Error bars indicate SD (n = 3). D, NHL1 depletion caused defects in kinetoplast segregation, cleavage furrow placement, and cytokinesis completion. Green: FAZ filament labeled with anti-CC2D antibody; pink: nuclear DNA and kinetoplast DNA stained with DAPI. The scale bar represents 5 μm. E, effects of NHL1 knockdown on kinetoplast segregation. Shown is the quantitation of binucleated cells with different configuration of the nucleus and the kinetoplast. Error bars indicate SD (n = 3). F, quantitation of dividing and nondividing cells. Error bars represent SD (n = 3). G, scanning electron microscopic analysis of dividing cells. The yellow arrowheads indicate the ingressing cleavage furrow. The scale bar represents 5 μm. DAPI, 4′,6-diamidino-2-phenylindole; FAZ, flagellum attachment zone; HA, hemagglutinin.

Figure 4

Depletion of NHL1 disrupts the positioning of the cell division plane.A, scanning electron microscopy to examine the cell division plane of dividing cells. The scale bar represents 5 μm. B, examination of the cell division plane of dividing cells using KLIF as a marker. Endogenous KLIF-3HA was detected by FITC-conjugated anti-HA antibody. Arrows indicate the KLIF-3HA signal that marks the cell division plane. The scale bar represents 5 μm. C, quantitation of dividing binucleated cells with normally positioned (blue) and mispositioned (red) cell division planes. Error bars represent SD (n = 3). D, measurement of the distance between the existing posterior and the nascent posterior of the dividing cells from noninduced control and NHL1 RNAi-induced cells. ∗∗∗p < 0.001 (Student’s t test). HA, hemagglutinin; NFD, new-flagellum daughter; OFD, old-flagellum daughter.

Figure 5

NHL1 is required for positioning of the new flagellum.A, anti-PFR2 immunostaining of the paraflagellar rod (PFR) within the flagella of binucleated cells. Arrows and arrowheads indicate the proximal ends of the new PFR and the old PFR, respectively. The scale bar represents 5 μm. B, anti-TbBILBO1 immunostaining of the flagellar pocket collar (FPC) of binucleated cells. Arrows and arrowheads indicate the new FPC and the old FPC, respectively. The scale bar represents 5 μm. C, 3D-SIM to examine the effect of NHL1 depletion on the segregation of the flagella and the FPCs, which were immunostained with the 20H5 antibody and the anti-TbBILBO1 antibody, respectively. Arrows and arrowheads indicate the new FPC and the old FPC, respectively. The scale bar represents 1 μm. D, measurement of the inter-FPC distance of binucleated cells. ∗∗∗p < 0.001 (Student’s t test). 3D-SIM, three-dimensional structured illumination microscopy. NF, new flagellum; OF, old flagellum.

Figure 6

Depletion of NHL1 disrupts basal body (BB) rotation and segregation.A, YL 1/2 and anti-TbSAS-6 immunostaining of the basal body of binucleated cells. Open arrowheads and arrows indicate new BB pair and old BB pair, respectively. The scale bar represents 5 μm. B, measurement of the interbasal body distance of binucleated cells. ∗∗∗p < 0.001 (Student’s t test). C, effect of NHL1 knockdown on basal body rotation and segregation. Anti-TbSAS-6 antibody labels basal body, and 20H5 antibody labels both the basal body and the flagellum. Open arrowheads and arrows indicate new BB pair and old BB pair, respectively. The scale bar represents 5 μm. The scale bar in the zoom-in image represents 1 μm. D, quantitation of binucleated cells with different basal body states. Error bars indicate SD (n = 3). mBB, mature basal body; NF, new flagellum; OF, old flagellum; pBB, probasal body.

Figure 7

Knockdown of NHL1 impairs hook complex segregation.A, immunofluorescence microscopic analysis of the centrin arm (CA) in binucleated cells. Anti-TbCentrin4/LdCen1 labels the CA and the BB, and YL 1/2 antibody labels the BB. Open arrowheads and arrows and indicate CA and BB, respectively. The scale bar represents 5 μm. B, measurement of inter-CA distance in binucleated cells. ∗∗∗p < 0.001 (Student’s t test). C, immunofluorescence microscopic analysis of the hook complex (HC) in binucleated cells. Anti-TbMORN1 labels the HC, and 20H5 labels the CA and the BB. Arrows and open arrowheads indicate the HC and the CA, respectively. The scale bar represents 5 μm. D, measurement of interhook complex distance in binucleated cells. ∗∗∗p < 0.001 (Student’s t test). BB, basal body.

Figure 8

NHL1 interacts with TbSpef1 and depends on TbSpef1 for localization to the MtQ.A, GST pull down to test the in vitro interaction between NHL1 and TbSpef1. Shown is a representative of three independent experiments. The input sample was 10% of the ∼475 μl cell lysate used for the pull-down of each bait protein. B, proximity ligation assay (PLA) to test the in vivo interaction between NHL1 and TbSpef1. Cells coexpressing PTP-NHL1 and TbSpef1-3HA were used. Mock: buffer only. Arrows indicate PLA signal. The scale bar represents 5 μm. C, quantitation of binucleate cells with different NHL1 fluorescence signal intensity. Error bars indicate SD (n = 3). D, effect of TbSpef1 knockdown on NHL1 localization to the MtQ. Endogenous PTP-NHL1 was detected by anti–protein A antibody. Open arrowheads and solid arrowheads indicate PTP-NHL1 signal in the new MtQ and the old MtQ, respectively. 20H5 was used to label the flagellum, the BB, and the CA. The scale bar represents 5 μm. E, effect of NHL1 depletion on the localization of TbSpef1 to the MtQ. Endogenous TbSpef1-3HA was detected with FITC-conjugated anti-HA antibody. Open arrowheads and solid arrowheads indicate TbSpef1-3HA signal in the new MtQ and the old MtQ, respectively. The scale bar represents 5 μm. BB, basal body; CA, centrin arm; CBB, Coomassie brilliant blue; HA, hemagglutinin; MtQ, microtubule quartet.