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Research Article
BOH1 cooperates with Polo-like kinase to regulate flagellum inheritance and cytokinesis initiation in Trypanosoma brucei
Kieu T. M. Pham, Qing Zhou, Yasuhiro Kurasawa, Ziyin Li
Journal of Cell Science 2019 132: jcs230581 doi: 10.1242/jcs.230581 Published 15 July 2019

ABSTRACT

Trypanosoma brucei possesses a motile flagellum that determines cell morphology and the cell division plane. Inheritance of the newly assembled flagellum during the cell cycle is controlled by the Polo-like kinase homolog TbPLK, which also regulates cytokinesis initiation. How TbPLK is targeted to its subcellular locations remains poorly understood. Here we report the trypanosome-specific protein BOH1 that cooperates with TbPLK to regulate flagellum inheritance and cytokinesis initiation in the procyclic form of T. brucei. BOH1 localizes to an unusual sub-domain in the flagellum-associated hook complex, bridging the hook complex, the centrin arm and the flagellum attachment zone. Depletion of BOH1 disrupts hook-complex morphology, inhibits centrin-arm elongation and abolishes flagellum attachment zone assembly, leading to flagellum mis-positioning and detachment. Further, BOH1 deficiency impairs the localization of TbPLK and the cytokinesis regulator CIF1 to the cytokinesis initiation site, providing a molecular mechanism for its role in cytokinesis initiation. These findings reveal the roles of BOH1 in maintaining hook-complex morphology and regulating flagellum inheritance, and establish BOH1 as an upstream regulator of the TbPLK-mediated cytokinesis regulatory pathway.

INTRODUCTION

Trypanosoma brucei, an early-branching unicellular protozoan, causes sleeping sickness in humans and nagana in animals in sub-Saharan Africa; it possesses a complex life cycle by alternating between the mammalian hosts and the tsetse fly vector. A T. brucei cell assembles a single flagellum that is necessary for cell locomotion, cell morphogenesis, cell division and cell–cell communication (Eliaz et al., 2017; Imhof et al., 2016; Kohl et al., 2003). The flagellum originates from the basal body located at the posterior part of the cell, exits the cell through the flagellar pocket and extends towards the anterior tip of the cell. The flagellum is attached, for the majority of its length, to the cell body through a cytoskeletal structure termed flagellum attachment zone (FAZ). The FAZ consists of multiple sub-domains located in the flagellum, the cell body and the junction between the flagellum and the cell body, and is composed of more than 20 proteins containing diverse structural motifs (Sunter and Gull, 2016). The FAZ is essential for flagellum attachment and basal body segregation and, together with the flagellum, controls cell division by defining the cell division plane and determining the site of cytokinesis initiation (Kohl et al., 2003; Zhou et al., 2011).

During the cell cycle T. brucei needs to precisely duplicate and segregate its multiple organelles and cytoskeletal structures in order to produce two identical daughter cells. 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., 2010, 2015, 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.

The bilobe structure in T. brucei, which was initially described as a novel centrin-labeled structure connecting the old and the new Golgi complex (He et al., 2005), was later found to display as a hairpin-like structure consisting of a TbMORN1-marked and TbLRRP1-marked fishhook-like structure – hereafter referred to as hook complex – and the TbCentrin4-marked arm – hereafter referred to as centrin arm (Esson et al., 2012; Morriswood, 2015). The hook part of the hook complex sits atop of the TbBILBO1-labeled flagellar pocket collar and the shank part of the hook complex; the centrin-arm flanks the FAZ filament and the quartet microtubules (Esson et al., 2012). The biological function(s) of this novel cytoskeletal structure remains to be fully explored, but work regarding the knockdown of TbLRRP1 in the procyclic form of T. brucei suggests that it plays an essential role in promoting the assembly of the new FAZ, thus, facilitating flagellum attachment (Zhou et al., 2010). TbLRRP1 depletion also inhibits cell division but this effect is likely to be attributed to the defective FAZ assembly and flagellum attachment, both of which are known to inhibit cytokinesis in T. brucei (LaCount et al., 2002; Zhou et al., 2011).

Cytokinesis in the procyclic (insect) form of T. brucei is initiated from the distal tip of the new FAZ, and requires a signaling cascade mediated by TbPLK, the Aurora B kinase homolog TbAUK1 and a cohort of trypanosome-specific proteins that act in concert at the new FAZ tip from early S phase to telophase of the cell cycle (Kumar and Wang, 2006; Kurasawa et al., 2018; Li et al., 2008; McAllaster et al., 2015; Zhou et al., 2016a,b; Zhou et al., 2018a). Among these cytokinesis regulators, the cytokinesis initiation factor 1 (CIF1) plays a pivotal role in recruiting multiple cytokinesis regulatory proteins to the new FAZ tip (Zhou et al., 2018a). It forms two separate protein complexes with cytokinesis initiation factors 2 and 3 (CIF2 and CIF3, respectively) and the two protein complexes function at the new FAZ tip to promote cytokinesis initiation (Kurasawa et al., 2018). CIF1 is recruited to the new FAZ tip by TbPLK and, in turn, exerts a feedback control on TbPLK by maintaining TbPLK at the new FAZ tip (Zhou et al., 2016a). TbPLK, thus, appears to be an upstream factor in the cytokinesis regulatory pathway; but how TbPLK is recruited to the new FAZ tip remains elusive.

In this paper, we report the identification of a hook complex-associated protein we named BOH1, which recruits TbPLK to the new FAZ tip such that TbPLK can further target CIF1 to promote cytokinesis initiation in the procyclic form of T. brucei. Additionally, depletion of BOH1 disrupts the morphology of the hook complex, inhibits elongation of the centrin arm, impairs FAZ elongation, and disrupts flagellum positioning and attachment. BOH1, thus, has multiple functions in promoting hook-complex morphogenesis, flagellum inheritance and cytokinesis initiation.

RESULTS

BOH1 localizes to an unusual sub-domain within the hook complex

Our previous BioID experiments with CIF1 as bait identified 15 proteins that localize to the hook complex (Zhou et al., 2018a,b). One of these proteins, encoded by Tb927.10.12720, localizes to a sub-domain in the hook complex that resembles a bar-shaped fishing bait hung on the hook part of the hook complex (Fig. 1). We, thus, named this protein bait on hook 1 (BOH1), and characterized its biological functions. BOH1 contains 12 coiled-coil motifs within amino acids (aa) 100–800, suggesting that it functions within a large protein complex. Using immunofluorescence microscopy, BOH1 was detected as a bar-shaped structure; one end (the posterior end) of the bar extended slightly beyond the hook part of the hook complex (Fig. 1A) and the other end (the distal end) of the bar connected to the centrin arm (Fig. 1B) and to the proximal end of the FAZ filament (Fig. 1C). BOH1 triply tagged with hemagglutinin (BOH1-3HA) displays a graded distribution of the fluorescence signal, with stronger signal intensity at the posterior than the distal end (Fig. 1A-C). During the S phase of the cell cycle, when the hook complex and the centrin arm start to duplicate (Fig. 1A-C), a new BOH1 fluorescence focus first emerges as a small dot near the hook part of the newly assembled hook complex (Fig. 1A). It barely overlaps with the newly formed centrin arm (Fig. 1B) and the newly assembled FAZ – i.e. the short new FAZ (snFAZ) – that has a length of ∼0.5–1.0 µm) (Fig. 1C). Following the cell cycle progression to late S phase, G2 phase and mitotic phases, the new BOH1 focus starts to overlap with the new centrin arm and the new FAZ (Fig. 1A-C).

Fig. 1.
Fig. 1.

BOH1 localizes to an unusual sub-domain of the hook complex. (A-C) DIC images of cells with different numbers of nuclei (N), kinetoplasts (K) or elongated kinetoplasts (eK). (A) BOH1 localization relative to the hook complex (HC). Shown are coimmunofluorescence microscopic images of BOH1-3HA (green) and of TbMORN1 (red) labeling the hook complex. Scale bar: 5 µm. (B) BOH1 localization relative to the centrin arm (CA). Shown are coimmunofluorescence microscopic images visualizing BOH1-3HA, and LdCen1 labeling the centrin arm. Scale bar: 5 µm. (C) BOH1 localizes to the proximal end of the FAZ filament. Shown are coimmunofluorescence microscopic images visualizing BOH1-3HA, and CC2D labeling the FAZ. Scale bar: 5 µm. d, day. (D,E) 3D-SIM super-resolution microscopic analysis of BOH1 localization relative to the hook complex and the FAZ filament. BOH1 was endogenously tagged with a triple HA epitope. Cells were coimmunostained with FITC-conjugated anti-HA monoclonal antibody and anti-TbMORN1 polyclonal antibody for hook complex (D) or FITC-conjugated anti-HA antibody and anti-CC2D polyclonal antibody for the FAZ filament (E). The small letters in the merged images indicate the viewing angles. Scale bars: 1 µm. (F) Schematic illustration of localization of BOH1 relative to the hook complex, the centrin arm, and the FAZ filament. In all images shown A-C, the cytoskeleton was used for immunofluorescence microscopy. BOH1-3HA was detected by FITC-conjugated anti-HA monoclonal antibody. TbMORN1, LdCen1 and CC2D were each detected by rabbit polyclonal antibodies. Old HC, old hook complex; New HC, new hook complex; Old FAZ, old FAZ later in the cell cycle; New FAZ, newly assembled FAZ from S phase onwards; Old CA, old centrin arm; New CA, new centrin arm; snFAZ, short new FAZ; FPC, flagellar pocket collar; mBB, mature basal body; pBB, pro-basal body.

To examine BOH1 localization at higher resolutions, we carried out super-resolution microscopy and examined the localization of BOH1 relative to the hook complex (Fig. 1D). The results showed that the posterior area of the BOH1 fluorescence signal is tilted towards the hook part of the hook complex, whereas its anterior area partly overlaps with the shank part of the hook complex (Fig. 1D). When viewed from the posterior end of the BOH1 signal, the posterior area of BOH1 wraps around the hook part of the hook complex (Fig. 1D,a). When viewed from the anterior end of the BOH1 signal, BOH1 is embedded within the hook complex (Fig. 1D,b). Viewing other angles of the coimmunofluorescence image confirmed the location of BOH1 within the hook complex (Fig. 1D,c-f). Further, we also examined the localization of BOH1 relative to the FAZ. The anterior area of the BOH1 signal is next to and partly overlaps with the proximal end of the FAZ (Fig. 1E). Viewing from different angles, coimmunofluorescence confirmed the relative location of BOH1 and the FAZ (Fig. 1E,a-d). Fig. 1F illustrates the location of BOH1 relative to hook complex, centrin arm and FAZ. Therefore, BOH1 might be component of a new sub-structure of the assembly between hook complex and centrin arm, which could play a role in anchoring the hook complex to the centrin arm and to the proximal end of the FAZ.

BOH1 is required for cytokinesis and flagellum inheritance

Tetracycline-inducible RNA interference (RNAi) was carried out to knock down BOH1 in the procyclic form of T. brucei to study its physiological function. To confirm the efficiency of RNAi, western blotting was performed to detect the protein level of BOH1-3HA. Induction of BOH1 RNAi resulted in rapid knockdown of BOH1 within one day, but complete depletion of BOH1 was not achieved even after 5 days (Fig. 2A). Proliferation of BOH1 RNAi cells was not affected during the first 2 days of RNAi but significantly slowed down after 3 days (Fig. 2B), indicating that BOH1 is essential for cell proliferation. To analyze the potential effect of BOH1 RNAi on cell cycle progression, we counted the cells with different numbers of kinetoplasts (K) and nuclei (N). The cell cycle stage for a T. brucei cell can be determined in a DAPI-stained cell by counting kinetoplasts and nuclei under a light microscope. Cells in G1 and S-phase contain one nucleus and one kinetoplast (1N1K), those in G2 and metaphase contain one nucleus and two kinetoplasts (1N2K), and those in anaphase, telophase and cytokinesis contain two nuclei and two kinetoplasts (2N2K). RNAi of BOH1 caused a significant decrease of 1N1K cells, a slight decrease of 1N2K cells and a slight increase of 2N2K cells (Fig. 2C). However, after 3 days of RNAi induction, some abnormal cell types – such as cells containing two nuclei and one kinetoplast (2N1K) and those containing no nucleus and one kinetoplast (0N1K) – emerged at ∼10% and ∼6%, respectively, (Fig. 2C). Notably, cells that contained multiple nuclei and multiple kinetoplasts (xNxK, x>2) increased to ∼50% of the total population (Fig. 2C). The increase in bi-nucleated (2N2K and 2N1K) and multi-nucleated (xNxK) cells in response to BOH1 RNAi suggests a defective cytokinesis.

Fig. 2.
Fig. 2.

BOH1 is required for flagellum positioning and attachment. (A) RNAi-mediated knockdown of BOH1 in the procyclic form of T. brucei. Shown is the western blot of BOH1 tagged with a triple HA epitope before and after RNAi induction. TbPSA6 served as a loading control. (B) RNAi of BOH1 caused a severe growth defect. (C) Effect of BOH1 knockdown on cell cycle progression. Shown is the quantification of cells with different numbers of nuclei (N) and kinetoplasts (K) after BOH1 RNAi. 200 cells were counted for each time point. Error bars indicate ±s.d. from three independent experiments (n=3). **P<0.01; ***P<0.001; snFAZ, short new FAZ. (D) BOH1 knockdown caused flagellum detachment. 200 cells were counted for each time point. Error bars indicate ±s.d. from three independent experiments (n=3). (E) Scanning electron microscopic images of control and BOH1 RNAi cells. NF, new flagellum; OF, old flagellum. Scale bars: 5 µm. (F) Immunofluorescence microscopic analysis of the flagellar pocket collar (FPC, arrows). Cells were treated with PEME buffer to prepare cytoskeleton and then immunostained with anti-TbBILBO1 antibody. Scale bar: 5 µm. (G) Quantification of inter-FPC distance in bi-nucleated cells. 100 cells for each cell type were used for measurement. ***P<0.001. (H) Immunofluorescence microscopic analysis of the basal body in control and BOH1 RNAi cells. Cells were treated with PEME buffer to prepare the cytoskeleton, and then immunostained with YL 1/2 antibody to label the mature basal body (mBB, arrows) and with anti-TbSAS-6 antibody to stain the mBB and the pro-basal body (pBB, arrowheads). Scale bar: 5 µm. (I) Quantification of inter-basal body distance in bi-nucleated cells. 100 cells for each cell type were used for measurement. ***P<0.001;

Another notable phenotype in BOH1 RNAi cells is the detachment of the new flagellum, which occurred from day 2 of RNAi in ∼10% of the cell population and gradually increased in ∼50% of the cell population after 6 days (Fig. 2D,E). There appeared to be no defect in the formation of the flagellum in BOH1 RNAi cells, as all the 2N2K and 2N1K cells contained two full-length flagella (Fig. 2E,F). The detached new flagellum appeared to be only slightly separated from the old flagellum (Fig. 2E and Fig. S1), suggesting that positioning of the new flagellum towards the cell posterior is likely to be impaired. To test this possibility, we examined the separation of two flagellum-associated cytoskeletal structures, the flagellar pocket collar (FPC), which was immunostained with the anti-TbBILBO1 antibody (Bonhivers et al., 2008), and the flagellar basal body (BB), which was labeled with the YL 1/2 antibody and the anti-TbSAS-6 antibody (Hu et al., 2015a). We focused on bi-nucleated cells, as the two FPCs and the two pairs of BB are fully separated in these cells under control conditions. In the control 2N2K cells, the two FPCs were separated on average by ∼5.5 µm, whereas in the BOH1-depleted 2N2K cells, ∼23% of them contained two FPCs that were separated on average by ∼2.1 µm and ∼75% of them contained two FPCs that were separated on average by ∼5.0 µm (Fig. 2F,G). There were a few cells (∼2%) in which the two PFCs were separated further (>7 µm) than in the control cells (Fig. 2G). In BOH1-depleted 2N1K cells, the average distance between the two FPCs was only ∼0.4 µm (Fig. 2F,G). Similarly, the two BB pairs in control 2N2K cells were separated on average by ∼5.5 µm, but in BOH1-deficient 2N2K cells, ∼24% of them contained two BB pairs that were separated on average by ∼2.5 µm and ∼74% of them contained two BB pairs that were separated on average by ∼4.6 µm (Fig. 2H,I). There were also a few cells (∼2%) containing two BB pairs spaced further apart (Fig. 2I) that, as is the case for cells comprising more-distant FPCs, were undergoing late stages of cytokinesis with two substantially separated daughter cells. Finally, in BOH1-depleted 2N1K cells, the two BB pairs were only separated on average by ∼0.9 µm (Fig. 2H,I). Together, these results demonstrate that BOH1 knockdown inhibited flagellum positioning and attachment.

Knockdown of BOH1 inhibits FAZ assembly and elongation

Previous work has shown that knockdown of the hook complex protein TbLRRP1 disrupts assembly of the new FAZ (Zhou et al., 2010). Depletion of BOH1 also caused flagellum detachment (Fig. 2D,E), suggesting that assembly of the new FAZ is also likely to be disrupted. To test this possibility, we performed immunofluorescence microscopy using anti-CC2D antibody to label the intracellular FAZ filament. T. brucei cells at the G1 phase (1N1K cells) contain a single FAZ (old FAZ later in the cell cycle), but they assemble a new FAZ from S phase, the cell cycle stage in which cells possess one nucleus and an elongated kinetoplast and (1N1eK). At this stage, some of the 1N1eK cells contain a short, new FAZ (snFAZ) and a full-length old FAZ (see Fig. 1C). Following cell cycle progression, the snFAZ further elongates to form a longer new FAZ in some 1N1eK cells and in all 1N2K and 2N2K cells (Fig. 3A). At days 1 and 2 of BOH1 RNAi, the majority (>90%) of cells lacked detached flagella (Fig. 2D), and there was also no defect in FAZ formation in these cells (Fig. S2). However, starting from day 3 of BOH1 RNAi, ∼17% of the cells had detached flagella (Fig. 2D); thus, we examined whether the FAZ is impaired in these cells. We found that the old FAZ filament was not affected by BOH1 depletion. However, the new FAZ filament in many of the bi-nucleated BOH1 RNAi cells (day 3) was significantly shorter than that in the control cells (Fig. 3A). Nonetheless, the new FAZ in these bi-nucleated RNAi cells (∼30%) had an average length of ∼2.0 µm, compared to the average length of ∼10 µm for the new FAZ in bi-nucleated control cells (Fig. 3B), suggesting that elongation of the new FAZ is impaired by BOH1 RNAi. Defective elongation of the new FAZ occurred in 1N1eK, 1N2K, 2N2K and abnormal 2N1K cells, in which the number of cells containing two FAZ filaments was significantly decreased (Fig. 3A,C). This was followed by a significant increase of 1N1eK cells containing a single FAZ, and of 1N2K, 2N2K and 2N1K cells containing either a single FAZ or an old FAZ plus snFAZ (Fig. 3A,C). Notably, ∼10% of 1N2K cells, ∼15% of 2N2K cells and ∼45% of 2N1K cells contained a single FAZ (Fig. 3C), suggesting that de novo assembly of a new FAZ was inhibited in these cells. Taken together, these results demonstrate that BOH1 is required for elongation of the new FAZ.

Fig. 3.
Fig. 3.

BOH1 is required for FAZ elongation. (A) DIC images of cells with different numbers of nuclei (N), kinetoplasts (K) or elongated kinetoplasts (eK). Immunofluorescence microscopic analysis of the FAZ in control and BOH1 RNAi cells. Cells were treated with cold methanol, and then immunostained with anti-CC2D polyclonal antibody to label the FAZ. snFAZ, short new FAZ; Old FAZ, old FAZ later in the cell cycle; New FAZ, newly assembled FAZ from S phase onwards; NF, new flagellum; OF, old flagellum. Scale bar: 5 µm. (B) BOH1 knockdown disrupted FAZ elongation. The length of the new FAZ filament in bi-nucleated cells was measured. 100 cells were used to measure the FAZ length. ***P<0.001. (C) Quantification of FAZ in control and BOH1 RNAi cells. 100 cells were counted for each cell type and for each time point. Error bars represent ±s.d. from three independent experiments (n=3). *P<0.05; **P<0.01; d, day.

BOH1 is required for maintaining the morphology of the hook complex and the centrin arm

Localization of BOH1 to the hook complex (Fig. 1) suggests that BOH1 plays a role in hook complex biogenesis and/or morphogenesis. Using the BOH1 RNAi cell line, we investigated the effect on the hook complex by immunofluorescence microscopy, using TbMORN1 and TbLRRP1-3HA as markers. In the majority of control cells, TbMORN1- and TbLRRP1-3HA-labeled hook complexes displayed the typical fishhook morphology (Fig. 4A,B), consistent with previous reports (Esson et al., 2012; Morriswood et al., 2009; Zhou et al., 2010). In some (∼14%) control cells, the hook complex appeared as a bar-shaped structure (Fig. 4C), probably because the structure was viewed at a different angle. In the majority of BOH1 RNAi cells, however, the hook complex seemed morphologically distorted (Fig. 4A,B). The hook complex either resembled an open hook, had a teardrop-like shape, was branched or elongated (Fig. 4B). Cells containing these morphologically abnormal hook complexes started to emerge at day 1 of BOH1 RNAi, followed by a significant increase for 2–3 days (Fig. 4C). This demonstrates that BOH1 is required to maintain hook-complex morphology.

Fig. 4.
Fig. 4.

Knockdown of BOH1 disrupts hook-complex morphology. (A) DIC images of cells with different numbers of nuclei (N) and kinetoplasts (K). Immunofluorescence microscopic analysis of the hook complex in control and BOH1 RNAi cells. Cytoskeleton was prepared for immunofluorescence microscopy. TbMORN1 was detected by anti-TbMORN1 antibody, TbLRRP1-3HA was detected by anti-HA antibody. Scale bar: 5 µm. (B) Schematic representation of the hook complex of various morphology in BOH1 RNAi cells. Scale bar: 1 µm. (C) Quantification of the hook complex of various morphology in control and BOH1 RNAi cells. 100 cells were counted for each time point. Error bars represent ±s.d. from three independent experiments (n=3). *P<0.05; **P<0.01; d, day.

We next examined whether the centrin arm was affected by BOH1 RNAi. The centrin arm can be marked by labeling centrin proteins that use the pan-centrin antibody 20H5 (He et al., 2005); but this antibody also labels the basal body. Therefore, to distinguish between basal body and the centrin arm, we coimmunostained cells with anti-TbSAS-6 antibody, which labels the cartwheel in the basal body (Hu et al., 2015a). In control cells, the centrin arm was detected as a small bar-shaped structure anterior to the basal body, with an average length of ∼0.9–1.1 µm (Fig. 5A,B). In BOH1 RNAi cells, however, the centrin arm was significantly shorter (Fig. 5A), with its average length calculated to be ∼0.4–0.5 µm (Fig. 5A,B). This result suggests that BOH1 is required for elongation of the centrin arm.

Fig. 5.
Fig. 5.

Depletion of BOH1 inhibits centrin arm elongation. (A) DIC images of cells with different numbers of nuclei (N) and kinetoplasts (K). Immunofluorescence microscopic analysis of the centrin arm (CA) by the pan-centrin antibody 20H5. Cytoskeleton was prepared for immunofluorescence microscopy. The basal body (BB) was labeled by anti-TbSAS-6 antibody. Scale bar: 5 µm. (B) Measurement of the centrin arm lengths in control and BOH1 RNAi cells. 100 cells for each cell type were used for measurement. ***P<0.001.

BOH1 interacts with and targets TbPLK to the bilobe structure, and the new FAZ tip

BOH1 has been identified as a proximal partner of CIF1 by proximity-dependent biotin identification (BioID) (Zhou et al., 2018a,b), but coimmunoprecipitation showed that the two proteins do not interact in vivo in T. brucei (Fig. S3). Since BOH1 plays a similar role as TbPLK in regulating flagellum positioning and attachment, and because TbPLK also localizes to the bilobe structure during early cell cycle stages (Ikeda and de Graffenried, 2012), we tested whether BOH1 and TbPLK interact in vivo. Coimmunoprecipitation showed that PTP-tagged TbPLK was able to pull down a small amount of BOH1-3HA protein from T. brucei cell lysate (Fig. 6A), suggesting that they interact in trypanosomes in vivo. We further carried out in vitro GST pull-down experiments to test the interaction between BOH1 and TbPLK. Owing to the insolubility of the full-length BOH1 protein in bacteria, we purified two truncation fragments of BOH1 (aa 1–281 and aa 282–576) as GST-fusion proteins from bacteria (Fig. S4) and used them for GST pull-down assays. The results showed that the N-terminal portion (aa 1–281) of BOH1 was able to pull down TbPLK from trypanosome cell lysate (Fig. 6B). The other fragment (aa 282–576) of BOH1 pulled down much less TbPLK (Fig. 6B). Immunofluorescence microscopy showed that, at G1 phase, TbPLK localized to the basal body and the hook complex (Hu et al., 2017b), where it colocalized with BOH1 (Fig. 6Ca). During early S phase, when BOH1 started to emerge at the new hook complex, TbPLK started to migrate from the hook complex to the newly assembled FAZ (snFAZ); thus, TbPLK only partly overlapped with BOH1 (Fig. 6Cb,c). During late S phase when the BOH1 protein on the new hook complex started to separate from the old hook complex, TbPLK was mainly localized to the new FAZ tip and, therefore, BOH1 and TbPLK did not overlap anymore (Fig. 6Cd). During G2 phase and early mitotic phases, TbPLK remained on the new FAZ tip and did not colocalize with BOH1, which remained on the hook complex (Fig. 6Ce,f). These results suggest that BOH1 and TbPLK colocalize in the assembly of hook complex and centrin arm during G1 phase, and partly colocalize during early S phase of the cell cycle.

Fig. 6.
Fig. 6.

BOH1 interacts with and colocalizes with TbPLK at the hook complex. (A) BOH1 interacts with TbPLK in vivo in trypanosomes. Coimmunoprecipitation was performed with IgG beads to pull down TbPLK-PTP and interacting BOH1-3HA, which was detected with anti-HA antibody. IB, immunoblotting. IP, immunoprecipitation. (B) BOH1 interacts with TbPLK in vitro, as demonstrated by GST pulldown assays. GST alone served as a negative control. (C) Coimmunofluorescence microscopic analysis of BOH1 and TbPLK during G1 and S phases of the cell cycle. BOH1 was endogenously tagged with a triple HA epitope and detected by FITC-conjugated anti-HA antibody. TbPLK was detected by anti-TbPLK polyclonal antibody. BB, basal body; HC, hook complex; snFAZ, short new FAZ; New FAZ tip, distal tip of the newly assembled FAZ from S phase onwards. Scale bar: 5 µm.

The in vivo interaction between BOH1 and TbPLK (Fig. 6A) prompted us to investigate the functional relationship between the two proteins. We first examined the effect of BOH1 depletion on TbPLK localization by immunofluorescence microscopy. TbPLK displays a dynamic subcellular localization during the cell cycle by localizing to the basal body and the bilobe structure in 1N1K cells, and to the new FAZ tip in 1N2K and 2N2K cells (Fig. 7A). Since BOH1 knockdown impaired elongation of new FAZ (Fig. 3) – which might indirectly affect TbPLK localization to the new FAZ tip – we carried out the experiments at early stages (day 2) of BOH1 knockdown and focused on the cells that did not have FAZ defects. Knockdown of BOH1 disrupted localization of TbPLK to the hook complex and basal body in 1N1K cells, and to the new FAZ tip in 1N2K and 2N2K cells (Fig. 7A,B). Western blotting showed that TbPLK protein level was not altered in BOH1 RNAi cells (Fig. 7C), indicating that TbPLK is likely to spread to the cytosol in BOH1-deficient cells. Together, these results demonstrate that BOH1 is required for TbPLK localization. We also investigated the effect of TbPLK depletion on BOH1 localization. Knockdown of TbPLK affected neither BOH1 localization (Fig. 7D) nor BOH1 protein level (Fig. 7E). Together, these results suggest that TbPLK functions downstream of BOH1.

Fig. 7.
Fig. 7.

BOH1 targets TbPLK to the hook complex and the new FAZ tip. (A) BOH1 RNAi abolished TbPLK localization to the hook complex and the new FAZ tip. TbPLK was detected with anti-TbPLK polyclonal antibody, and FAZ1 was labeled with anti-FAZ1 antibody. BB, basal body; HC, hook complex; New FAZ tip, distal tip of the newly assembled FAZ from S phase onwards. Scale bar: 5 µm. (B) Quantification of TbPLK localization in control and BOH1 RNAi cells. 100 cells were counted for each time point and each cell type. Error bars indicate ±s.d. from three independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001. (C) Effect of BOH1 knockdown on TbPLK protein level. TbPLK was detected by anti-TbPLK antibody, BOH1-3HA by anti-HA antibody and TbPSA6 by anti-TbPSA6 antibody. (D) DIC images of cells with different numbers of nuclei (N) and kinetoplasts (K). Effect of TbPLK knockdown on BOH1 localization. BOH1-3HA (arrowheads) was detected by FITC-conjugated anti-HA antibody, and TbPLK by anti-TbPLK antibody. Scale bar: 5 µm. (E) Western blotting to monitor BOH1 protein level in TbPLK RNAi cells. BOH1-3HA was detected by anti-HA antibody, TbPLK by anti-TbPLK antibody and TbPSA6 by anti-TbPSA6 antibody.

BOH1 is required for the localization of the cytokinesis regulator CIF1 to the new FAZ tip

Depletion of BOH1 caused a significant increase of multi-nucleated (xNxK) cells (Fig. 2C), indicating defective cytokinesis. It is noteworthy that the generation of multi-nucleated cells appeared to coincide with the accumulation of cells with detached flagella (Fig. 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., 2015, 2011). Nonetheless, our finding that BOH1 deficiency disrupted TbPLK localization to the FAZ tip (Fig. 7A, B) implies that BOH1 plays an essential role in cytokinesis. To further corroborate this result, we examined the effect of BOH1 depletion on the localization of CIF1, a substrate of TbPLK and an essential regulator of cytokinesis initiation in T. brucei (Zhou et al., 2016a). We focused on cells at the early stage (day 2) of BOH1 RNAi – at this time point, assembly of the new FAZ was not impaired (Fig. S2) – to avoid potential pleiotropic effects due to the defects on flagellum-associated structures (Figs 3–5). By using anti-CIF1 polyclonal antibody for immunofluorescence microscopy, CIF1 was detected at the new FAZ tip in S-phase (1N1eK) cells, G2 (1N2K) cells, and mitotic (2N2K) cells, but not G1 (1N1K) cells under control conditions (Fig. 8A). In BOH1 RNAi cells, however, CIF1 localization was impaired (Fig. 8A,B). Among the 1N1eK and 1N2K cells, this number of cells in which CIF1 localized to the new FAZ tip, was significantly decreased. This was followed by the emergence of cells, in which CIF1 localized to the flagellar pocket region or to both the new FAZ tip and the flagellar pocket region (Fig. 8A,B). In 2N2K cells, similar patterns of CIF1 localization were observed, except that the number of cells without any CIF1 signals was significantly increased (Fig. 8A,B). Western blotting showed that CIF1 protein levels were not altered in BOH1 RNAi cells (Fig. 8C), indicating that CIF1 is likely to spread to the cytosol in those 2N2K cells that lack detectable CIF1 signal. To rule out the possibility that the disruption of the hook complex affects CIF1 localization indirectly, we examined the location of CIF1 in TbLRRP1 RNAi cells, which are known to have defects in the hook complex (Zhou et al., 2010). Our results showed that depletion of TbLRRP1 did not affect CIF1 localization to the new FAZ tip (Fig. S5), suggesting that the effect of BOH1 RNAi on CIF1 localization is due to BOH1 depletion but not to defects of the hook complex.

Fig. 8.
Fig. 8.

BOH1 is required for localization of the cytokinesis regulator CIF1 to the new FAZ tip. (A) Immunofluorescence microscopic analysis of CIF1 localization in control and BOH1 RNAi cells. Cells were treated with PEME to prepare cytoskeleton for immunofluorescence microscopy. CIF1 was stained with anti-CIF1 antibody, and the FAZ filament was labeled with anti-FAZ1 antibody. FP, flagellar pocket; New FAZ tip, distal tip of the newly assembled FAZ from S phase onwards. Scale bar: 5 µm. (B) Quantification of CIF1 localization in control and BOH1 RNAi cells. 100 cells were counted for each cell type and for each time point. Error bars indicate ±s.d. from three independent experiments (n=3). ***P<0.001. (C) CIF1 protein levels in control and BOH1 RNAi cells. CIF1 was detected by anti-CIF1 antibody, BOH1-3HA by anti-HA antibody and TbPSA6 by anti-TbPSA6 antibody. (D) Effect of CIF1 RNAi on BOH1 localization. Cytoskeletons were prepared for immunofluorescence microscopy. BOH1-3HA was immunostained with FITC-conjugated anti-HA antibody, and CIF1 was detected by anti-CIF1 antibody. Scale bar: 5 µm. (E) BOH1 protein levels in CIF1 RNAi cells. BOH1-3HA was detected by anti-HA antibody, CIF1 was detected by anti-CIF1 antibody, and TbPSA6 by anti-TbPSA6 antibody.

Finally, we tested whether knockdown of CIF1 exerted any effect on BOH1 localization. Depletion of CIF1 by RNAi affected neither the localization of BOH1to the hook complex nor the level of BOH1 protein (Fig. 8D,E), demonstrating that CIF1 acts downstream of BOH1 in the cytokinesis regulatory pathway. These results also demonstrate an essential role of BOH1 in promoting cytokinesis initiation.

DISCUSSION

In this article, we identified the trypanosome-specific protein BOH1. It localizes to a previously unidentified sub-domain in the structure comprising the hook complex and the centrin arm, bridging hook complex, centrin arm and FAZ filament (Figs 1 and 2), and is required for morphogenesis of the hook complex and centrin arm, and FAZ assembly and cytokinesis initiation (Figs 2–8). Knockdown of BOH1 disrupted the typical fishhook shape of the hook complex (Fig. 4), suggesting that BOH1 is necessary to maintain the framework of the hook complex. However, the precise role of the hook complex remains elusive. The hook complex is defined by TbMORN1 and TbLRRP1, and depletion of the two proteins yields distinct defects (Morriswood et al., 2009; Zhou et al., 2010). TbMORN1 is not essential in the procyclic form but required for protein entry to the flagellar pocket and cell viability in the bloodstream form (Morriswood et al., 2009; Morriswood and Schmidt, 2015). TbLRRP1, however, is required for biogenesis of the new FAZ and for cell viability in the procyclic form (Zhou et al., 2010). It appears that the hook complex does not act as an entity for any specific cellular function(s), although its components might play distinct roles in diverse cellular processes. Depletion of BOH1 inhibited elongation of the centrin arm (Fig. 5); however, consequences of this defect are still unclear. This is mainly due to the lack of a well-defined role of the centrin arm. Only two proteins, TbCentrin4 and TbCentrin2 (He et al., 2005; Shi et al., 2008), have so far been found to localize to the centrin arm, and both have been functionally characterized (He et al., 2005; Selvapandiyan et al., 2007; Shi et al., 2008). However, the phenotypic defects caused by genetic ablation of the two centrin proteins are complicated because they localize to the basal body as well as the centrin arm, making a dissection of the role of the centrin arm impossible. Although the mechanistic role of BOH1 in bilobe morphogenesis remains to be determined, we speculate – on the basis of its unusual localization (Fig. 1), and the RNAi-induced defects in the morphology of the hook complex and the centrin arm (Figs 4 and 5) – that BOH1 functions as an anchor to retain the relative position of the hook complex and the centrin arm, thereby maintaining the typical morphology of both structures.

The defect in flagellum attachment and positioning caused by BOH1 RNAi (Fig. 2) is attributed to the inhibited assembly/elongation of the new FAZ (Fig. 3). This conclusion is based on the fact that proper assembly and elongation of the new FAZ not only mediates flagellum adhesion (LaCount et al., 2002; Vaughan et al., 2008; Zhou et al., 2015, 2011), but also controls the positioning of the new flagellum and the new basal body (Absalon et al., 2007). Given that BOH1 knockdown generated some cells containing only an old FAZ (Fig. 3C), it suggests that assembly of the new FAZ was completely abolished in these cells. Thus, BOH1 appears to promote the de novo assembly of the new FAZ. Furthermore, given that BOH1 knockdown also generated some cells containing an snFAZ and an old FAZ (Fig. 3C), it suggests that BOH1 also plays a role in promoting the elongation of the new FAZ. The effect of BOH1 RNAi on FAZ assembly and elongation is similar to that caused by TbLRRP1 RNAi (Zhou et al., 2010), but differs from that caused by RNAi of FAZ filament proteins, such as CC2D and FAZ2, which generated cells containing an snFAZ and an old FAZ, but not cells containing only an old FAZ (Zhou et al., 2015, 2011). These results suggest distinct roles between hook complex-associated proteins and FAZ filament proteins in regulating the assembly of the new FAZ. Previous reports demonstrated that assembly of the new FAZ occurs at its proximal end (Sunter et al., 2015; Zhou et al., 2015), and the proximal end of the FAZ is tethered between the centrin arm and the shank part of the hook complex (Esson et al., 2012). These findings provided the structural basis for the involvement of the structure comprising the hook complex and the centrin arm in FAZ assembly. Moreover, the similarity in function but the distinction in localization within the hook complex between BOH1 and TbLRRP1 suggests that it is likely to be the hook complex per se that dictates the de novo assembly and elongation of the new FAZ. In this regard, BOH1 and TbLRRP1 might have similar functions in maintaining the integrity of the structure comprising hook complex and centrin arm.

There is considerable evidence to support the notion that BOH1 plays an essential role in promoting initiation of cytokinesis in the procyclic form of T. brucei. First, BOH1 interacts with and targets TbPLK to the new FAZ tip (Figs 6 and 7). TbPLK is a key cytokinesis regulator that acts most upstream of the cytokinesis regulatory pathway, involving Aurora B kinase TbAUK1, and the three trypanosome-specific cytokinesis regulators CIF1, CIF2 and CIF3 (Kurasawa et al., 2018; Zhou et al., 2018a, 2016a,b). Second, BOH1 is required for localization of CIF1 to the new FAZ tip (Fig. 8), and our previous results demonstrate that localization of CIF1 to the new FAZ tip is essential for initiation of cytokinesis (Hu et al., 2017a). The finding that BOH1 is required for TbPLK localization but not vice versa (Fig. 7) places BOH1 upstream of TbPLK in the cytokinesis regulatory pathway. Although precisely how BOH1 targets TbPLK remains unclear, it is possible that BOH1 serves as a docking site on the hook complex to recruit TbPLK during early cell cycle stages, such as G1 and S. Given that BOH1 also functions to maintain the structural integrity of the hook complex and the centrin arm (Figs 4 and 5), it appears that BOH1 has multiple functions.

BOH1 is probably the first hook complex-localizing protein found to play an essential role in the initiation of cytokinesis through regulating key cytokinesis regulators. It has previously been shown that depletion of the hook complex-localizing protein TbLRRP1 also inhibited cell proliferation (Zhou et al., 2010), but the observed defective cell division can probably be attributed to defects in FAZ assembly and flagellum attachment, which are known to have a pleiotropic effect on cell division (LaCount et al., 2002; Zhou et al., 2011). Depletion of TbLRRP1 did not affect localization of the cytokinesis regulator CIF1 to the new FAZ tip (Fig. S5), thus, distinguishing it from BOH1 in the regulation of cytokinesis regulator(s). Another report showed that depletion of TbSmee1, which localizes to the shank part of the hook complex and the new FAZ tip, disrupts localization of TbPLK to the new FAZ tip and inhibits cytokinesis (Perry et al., 2018). However, since TbSmee1 shows dual location, its role in regulating cytokinesis may be attributed to its function at the new FAZ tip, whereas that in regulating hook-complex morphogenesis may be linked to its function at the shank part of the hook complex. Given the functional distinctions between these hook complex-associated proteins, we suggest that the specific role of BOH1 in regulating initiation of cytokinesis is attributed to its localization to an unusual sub-domain in the hook complex (Fig. 1), where it interacts with TbPLK (Fig. 6). It will be interesting to investigate whether any proteins other than BOH1 also localize to the same unusual sub-domain of the hook complex and whether these proteins regulate cytokinesis initiation. This will help to understand the roles of the hook complex in initiation of cytokinesis.

MATERIALS AND METHODS

Trypanosome cell culture

Cells used in this study were derivatives of the procyclic form of T. brucei Lister 427 strain (ATCC, NR-42010), which has been recently authenticated and tested for contamination. The wild-type Lister 427 strain was cultured in the SDM-79 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) at 27°C. The 29-13 strain, which expresses the T7 RNA polymerase and the tetracycline repressor protein (ATCC, NR-42012) (Wirtz et al., 1999), was cultured in the SDM-79 medium containing 10% FBS plus 15 µg/ml G418 and 50 µg/ml hygromycin. Cells were sub-cultured routinely by diluting them 10-fold with fresh medium once the cell density had reached 5×106 cells/ml.

Endogenous epitope tagging of proteins

Endogenous tagging of BOH1 and TbLRRP1 with a C-terminal triple hemagglutinin (HA) epitope at one of their endogenous loci was carried out using the PCR-based one-step epitope-tagging approach (Shen et al., 2001). PCR products were purified from agarose gel and electroporated into the 427 strain and the BOH1 RNAi cell line. Transfectants were selected with 1 µg/ml puromycin, and then cloned by limiting dilution into a 96-well plate containing SDM-79 medium supplemented with 20% heat-inactivated FBS and appropriate antibiotics.

For coimmunoprecipitation of TbPLK and BOH1, TbPLK was tagged with a C-terminal PTP epitope in the 427 strain expressing BOH1-3HA, using the PCR-based method described above. Transfectants were selected with 40 µg/ml G418 in addition to 1.0 µg/ml puromycin, and were further cloned by limiting dilution as described above.

RNA interference

To generate the BOH1 RNAi cell line, a 566-bp DNA fragment (nucleotide 1139–1704) from the coding region of the BOH1 gene was cloned into the pZJM vector (Wang et al., 2000). The resulting plasmid, pZJM-BOH1, was linearized with NotI and transfected into cells of the 29-13 strain by electroporation. Transfectants were selected with 2.5 µg/ml phleomycin and, subsequently, cloned by limiting dilution as described above. RNAi was induced with 1.0 µg/ml tetracycline, and cell growth was monitored by daily counting of cells with a hemacytometer under a light microscope. The TbPLK RNAi cell line (Zhou et al., 2016a) and the TbLRRP1 RNAi cell line (Zhou et al., 2010) have been described previously. Efficiency of RNAi was monitored by western blotting, using anti-TbPLK polyclonal antibody for TbPLK RNAi cells, anti-HA monoclonal antibody for BOH1-3HA for BOH1 RNAi cells and TbLRRP1-3HA for TbLRRP1 RNAi cells. The anti-TbPSA6 polyclonal antibody, which detects the α-6 subunit of the 26S proteasome (Li et al., 2002), was used as a loading control.

Immunofluorescence microscopy

Cells were collected by centrifugation, washed once with phosphate-buffered saline (PBS) and adhered onto glass coverslips at room temperature for 30 min. Cells were fixed with cold methanol (−20°C) for 30 min. Cytoskeletons were prepared by treating cells with PEME buffer (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA, 1 mM MgSO4) supplemented with 0.1% Nonidet-P40 at room temperature for 5 min. The fixed cells or cytoskeleton samples were incubated with 3% bovine serum albumin (BSA) in PBS for 20 min at room temperature. Immunostaining was performed by incubating fixed cells with a primary antibody for 1 h at room temperature. Primary antibodies used were: fluorescein isothiocyanate (FITC)-conjugated anti HA monoclonal antibody (Clone HA-7, H7411, Sigma-Aldrich, 1:400 dilution), anti-TbMORN1 polyclonal antibody (1:400 dilution) (Morriswood et al., 2009), anti-LdCen1 polyclonal antibody (1:1000 dilution) (Selvapandiyan et al., 2007), anti-TbBILBO1 polyclonal antibody (1:400 dilution) (Bonhivers et al., 2008), anti-CC2D polyclonal antibody (1:400 dilution) (Zhou et al., 2011), anti-TbSAS6 polyclonal antibody (1:1000 dilution) (Hu et al., 2015a), 20H5 monoclonal antibody (EMD-Millipore, 04-1624, 1:400 dilution) (Lingle and Salisbury, 1997), L3B2 (anti-FAZ1) monoclonal antibody (1:5 dilution) (Kohl et al., 1999), YL1/2 monoclonal antibody (EMD Millipore, MAB1864, 1:400 dilution) (Kilmartin et al., 1982), anti-CIF1 polyclonal antibody (1:400 dilution) (Zhou et al., 2018a), and anti-TbPLK polyclonal antibody (1:400 dilution) (Hu et al., 2015b). After washing cells on the coverslip three times with PBS, they were incubated with a secondary antibody, such as FITC-conjugated anti-rat IgG (Sigma-Aldrich, F6258, 1:400 dilution), FITC-conjugated anti-mouse IgG (Sigma-Aldrich, F0257, 1:400 dilution), Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, AP132C, 1:400 dilution), and Cy3-conjugated anti-mouse IgG (Sigma-Aldrich, AP124C, 1:400 dilution), at room temperature for 1 h. Cells were then washed three times with PBS and one time with distilled water before permanently mounting them in DAPI-containing VectaShield mounting medium (Vector Lab). Cells were observed with an inverted fluorescence microscope (Olympus IX71) equipped with a cooled CCD camera (Hamamatsu, Japan) and a PlanApo N 60X1.42 NA oil lens. Images were acquired by using the Slidebook 5 software.

3D-structured illumination microscope (3D-SIM) super-resolution microscopy

Cells were adhered to the No. 1.5 high-precision glass coverslip, and then treated with PEME buffer containing 1% NP-40 for 1 min. Cells were fixed in cold methanol (−20°C) for 30 min and then incubated in blocking buffer (1% BSA in PBS). Cells were coimmunostained with FITC-conjugated anti-HA monoclonal antibody (Clone HA-7, H7411, Sigma-Aldrich, 1:400 dilution) and anti-TbMORN1 polyclonal antibody (1:400 dilution) (Morriswood et al., 2009) or anti-CC2D polyclonal antibody (1:400 dilution) (Zhou et al., 2011). Cells were washed three times with PBS, and then incubated with Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, AP132C, 1:400 dilution). Slides were viewed under Nikon Super Resolution Microscope n-SIM E (Nikon Instruments Inc., Americas). The acquired SIM images were applied to Stack 3D-structured illumination microscope (3D-SIM) reconstruction and analyzed by using NIS-Elements AR software.

Scanning electron microscopy

Sample preparation for scanning electron microscopy was performed according to the method described in our previous publication (Zhou et al., 2016a). Cells were settled onto glass coverslips and fixed with 2.5% (v/v) glutaraldehyde in PBS for 30 min in the dark at room temperature. Cells were then dehydrated by treating washes in a series of alcohol (30%, 50%, 70%, 90% and 100%) for 5 min each at room temperature. After critical point drying, coverslips were coated with a 8-nm metal film (Pt:Pd 80:20, Ted Pella Inc.) by using a sputter coater (Cressington Sputter Coated 208 HR, Ted Pella Inc.) and examined by using Nova NanoSEM 230 (FEI). Parameters used were 5 mm for the scanning work distance and 8 kV for the accelerating high voltage.

GST pull-down assays and coimmunoprecipitation

For GST pull-down experiments, two truncation fragments of BOH1 (aa 1–281 and aa 282–576) were fused with an N-terminal GST tag by cloning their corresponding DNA fragments into the pGEX-4T-3 vector (Clontech). The resulting plasmids were each transformed into E. coli BL21 strain and recombinant proteins were purified by passing the cell lysate through a glutathione sepharose 4B column (GE HelathCare). GST pull-down assays were carried out as described previously (Zhou et al., 2018a).

The trypanosome cell line expressing 3HA-tagged BOH1 and PTP-tagged TbPLK was washed once with PBS, re-suspended in 500 µl immunoprecipitation buffer (25 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM DTT, 1% NP-40 and protease inhibitor cocktail). Cells were lysed by sonication, and cell lysate was cleared by centrifugation. The supernatant was incubated with 30 µl settled IgG Sepharose beads (GE HealthCare) for 1 h at 4°C with gentle rotation. IgG beads were washed five times with immunoprecipitation buffer; bound proteins were eluted with 30 µl 10% SDS. Immunoprecipitated proteins were separated by SDS-PAGE, transferred onto a PVDF membrane and immunoblotted with anti-Protein A polyclonal antibody (1:2000 dilution, Sigma-Aldrich, P3775) and anti-HA antibody (1:5000 dilution, Sigma-Aldrich, Clone HA-7, H9658) to detect TbPLK-PTP and BOH1-3HA, respectively.

Data analysis and statistical analysis

ImageJ software (National Institutes of Health, Bethesda, MD; http://imagej.nih.gov/ij/) was used to measure the length of new FAZ, the length of the centrin arm, the inter-basal body distance and the inter-flagellar pocket collar distance. Data were exported to Microsoft Excel and GrapPad Prism5 for analysis. Statistical analysis was conducted using Student's t-test in Microsoft Excel and GraphPad Prism5. Error bars represent the standard deviation (±s.d.) of the mean from three biological independent replications. For immunofluorescence microscopy experiments, images were taken randomly and all images were used for analysis.

Acknowledgements

We thank Dr Cynthia Y. He (National University of Singapore, Singapore) for providing the anti-CC2D antibody, Dr Brooke Morriswood (University of Würzburg, Würzburg, Germany) for providing the anti-TbMORN1 antibody, and Dr Hira Nakashi (U.S. Food and Drug Administration, Washington, D.C.) for providing the anti-LdCen1 antibody. Gratitude also goes to Dr James Gu (Houston Methodist Research Institute, Houston, TX) for his assistance with scanning electron microscopy.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    Conceptualization: Z.L.; Methodology: K.T.M.P., Q.Z., Y.K.; Validation: K.T.M.P., Q.Z., Y.K.; Formal analysis: K.T.M.P., Z.L.; Investigation: K.T.M.P., Q.Z., Y.K.; Writing - original draft: Z.L.; Writing - review & editing: Z.L.; Visualization: K.T.M.P., Q.Z., Y.K.; Supervision: Z.L.; Project administration: Z.L.; Funding acquisition: Z.L.

  • Funding

    This work was supported by R01 grants from the National Institutes of Health (grant numbers: AI101437 and AI118736 to Z.L.). Deposited in PMC for release after 12 months.

  • Supplementary information

    Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.230581.supplemental

  • Received February 1, 2019.
  • Accepted June 11, 2019.

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Research Article
BOH1 cooperates with Polo-like kinase to regulate flagellum inheritance and cytokinesis initiation in Trypanosoma brucei
Kieu T. M. Pham, Qing Zhou, Yasuhiro Kurasawa, Ziyin Li
Journal of Cell Science 2019 132: jcs230581 doi: 10.1242/jcs.230581 Published 15 July 2019
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