Polo-like kinase phosphorylation of the orphan kinesin KIN-G negatively regulates centrin arm biogenesis in Trypanosoma brucei
- Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center at Houston, United States
- Department of Tropical Medicine, College of Medicine, Soon Chun Hyang University, Republic of Korea
Figures
Figure 1
KIN-G is a substrate of TbPLK in T. brucei.
(A) Co-immunostaining of KIN-G-3HA and TbPLK during the cell cycle in procyclic form. Open arrowheads indicate KIN-G signal at the centrin arm. TbPLK signal at different structures is indicated. BB: basal body; CA: centrin arm; FC: flagella connector; FAZt: FAZ tip. Scale bars: 5 μm. (B) TbPLK phosphorylates KIN-G in vitro. The asterisk indicates a nonspecific band. (C) In vitro phosphorylated KIN-G migrates slower than non-phosphorylated KIN-G on SDS-PAGE. (D) Co-immunoprecipitation of native KIN-G protein by WDR2-3HA from cells treated with or without GW843682X and from cell lysate treated with Lambda protein phosphatase (λPPase). Shown is a silver-stained 6% SDS-PAGE gel. (E) In vitro TbPLK phosphosites on KIN-G protein identified by mass spectrometry. Phosphosites highlighted in red indicate the in vitro and in vivo phosphosites. MD: motor domain; CC: coiled coil; MB: microtubule-binding motif; NB: nucleotide-binding motif. (F) Phosphosites within the KIN-G protein sequence spanning MB1 to MB3. Sequences highlighted in blue indicate the microtubule-binding motifs (MB1, MB2, and MB3), and sequences highlighted in green indicate the nucleotide-binding motifs (NB2 and NB3). (G) Effect of GW843682X treatment on the phosphorylation levels of Thr301 and Ser569. Shown is the % of reduction of phosphorylated peptides after GW843682X treatment. Error bars indicate SD from three independent experiments. (H) Percentage of phosphosites and non-phosphosites of Thr301- and Ser569-containing peptides in wild-type trypanosome cells. Error bars indicate SD from three independent experiments.
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Figure 1—source data 1
PDF file containing original western blots, Coomassie blue-stained gel image, and silver-stained gel image for Figure 1B–D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110793/elife-110793-fig1-data1-v1.pdf
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Figure 1—source data 2
Original files for western blot analysis, Coomassie blue staining, and silver staining displayed in Figure 1B–D.
- https://cdn.elifesciences.org/articles/110793/elife-110793-fig1-data2-v1.zip
Figure 2 with 4 supplements
Phosphorylation of KIN-G by TbPLK disrupts the microtubule-binding activity of KIN-G.
(A) Pre-incubation of KIN-G with TbPLK, but not TbPLKK70R, disrupted KIN-G microtubule-binding activity. KIN-G, KIN-G and TbPLK mixture, and KIN-G and TbPLKK70R mixture were first attached to coverslips and then microtubules (MTs) were added into the chamber. Scale bar: 10 µm. (B) Quantitation of KIN-G-bound microtubules in the presence or absence of TbPLK or in the presence of TbPLKK70R. Error bars indicate SD from three independent experiments. ****: p<0.0001; ns: no significance (one-way ANOVA). (C) Measurement of the microtubule-gliding speed of KIN-G in the presence or absence of TbPLK or TbPLKK70R. Error bars indicate SD from three independent experiments. ND: not done. ns: no significance (one-way ANOVA). (D) TbPLK disrupted KIN-G microtubule-binding activity. KIN-G was first attached to coverslips, MTs were added into the chamber, and then TbPLK was added into the chamber. Scale bar: 10 µm. (E) Quantitation of KIN-G-bound microtubules following the incubation with or without TbPLK. Error bars indicate SD from three independent experiments. **: p<0.01; ***: p<0.001 (Student’s t-test).
Figure 2—video 1
Microtubule-binding and -gliding activities of KIN-G.
Scale bar: 10 µm.
Figure 2—video 2
Microtubule-binding and -gliding activities of KIN-G, which was pre-incubated with TbPLK.
Scale bar: 10 µm.
Figure 2—video 3
Microtubule-binding and -gliding activities of KIN-G, which was pre-incubated with the kinase-dead mutant TbPLKK70R.
Scale bar: 10 µm.
Figure 2—video 4
Microtubule-binding and -gliding activities of KIN-G, with TbPLK added to the chamber after microtubules were added.
Scale bar: 10 µm.
Figure 3 with 5 supplements
Phosphorylation of Thr301 on KIN-G by TbPLK disrupts the microtubule-binding activity of KIN-G.
(A) Microtubule-binding activity of KIN-G and its Thr284 and Thr301 mutants. Scale bar: 10 µm. (B) Quantitation of the bound microtubules of KIN-G and its mutants. Error bars indicate SD from three independent experiments. **: p<0.01; ns: no significance (one-way ANOVA). (C) Measurement of the microtubule-gliding speed of KIN-G and its mutants. Error bars indicate SD from three independent experiments. ND: not done. ns: no significance (one-way ANOVA).
Figure 3—video 1
Microtubule-binding and -gliding activities of KIN-G.
Scale bar: 10 µm.
Figure 3—video 2
Microtubule-binding and -gliding activities of KIN-GT301D.
Scale bar: 10 µm.
Figure 3—video 3
Microtubule-binding and -gliding activities of KIN-GT301A.
Scale bar: 10 µm.
Figure 3—video 4
Microtubule-binding and -gliding activities of KIN-GT284D.
Scale bar: 10 µm.
Figure 3—video 5
Microtubule-binding and -gliding activities of KIN-GT284A.
Scale bar: 10 µm.
Figure 4
Expression of Thr301 phospho-mimic mutant of KIN-G disrupts cell proliferation.
(A) Subcellular localization of ectopically expressed KIN-G, KIN-GT301A, KIN-GT301D, co-stained with TbCentrin4. BB: basal body; CA: centrin arm. Scale bar: 5 μm. (B) Western blotting to detect the levels of ectopically 3HA-tagged KIN-G and its mutants and the endogenously PTP-tagged KIN-G before and after tetracycline induction for 48 hr. TbPSA6 serves as loading control. (C) Growth curves of KIN-G RNAi cell line and its complementation cell lines expressing KIN-G, KIN-GT301A, or KIN-GT301D. OE: overexpression. Error bars indicate SD from three independent experiments. (D) Quantitation of the numbers of nuclei (N) and kinetoplasts (K) of KIN-G RNAi cell line and its complementation cell lines. 100 cells were counted for each time point, and error bars indicated SD from three independent experiments.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110793/elife-110793-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/110793/elife-110793-fig4-data2-v1.zip
Figure 5
TbPLK phosphorylation of Thr301 on KIN-G disrupts centrin arm and Golgi biogenesis.
(A) Effect of the expression of KIN-GT301D on centrin arm formation. Cells were co-immunostained with the pan-centrin antibody 20H5 and the anti-TbCentrin4 antibody. nCA: new centrin arm; oCA: old centrin arm. Scale bar: 5 μm. (B) Measurement of centrin arm length in KIN-G RNAi cells expressing Thr301 phospho-mimic of KIN-G before and after tetracycline induction. 100 cells were used for measurement for each time point, and error bars indicated SD. CA: centrin arm. ns: no significance; ****: p<0.0001 (Student’s t-test). (C) Effect of the expression of KIN-GT301D on the biogenesis of Golgi and endoplasmic reticulum exit site (ERES). Cells were co-immunostained with the anti-TbGRASP antibody to detect TbGRASP. ERES was labeled by mCherry-tagged Sec13. Scale bar: 5 μm. (D) Quantitation of cells with different numbers of Golgi/ERES in control and KIN-G RNAi cells. 100 cells were counted for each time point, and error bars indicated SD from three independent experiments. ***: p<0.001; ****: p<0.0001 (one-way ANOVA).
Figure 6
TbPLK phosphorylation of Thr301 on KIN-G impairs FAZ elongation and flagellar positioining.
(A) Effect of the expression of KIN-GT301D on the elongation of the flagellum attachment zone (FAZ) filament. Cells were immunostained with the anti-CC2D antibody. nFAZ: new FAZ; oFAZ: old FAZ. Scale bar: 5 μm. (B) Measurement of the length of the new and the old FAZ filaments in control and KIN-G RNAi cells expressing KIN-GT301D. 100 cells were used for measurement for each time point, and error bars indicated SD. **: p<0.01; ****: p<0.0001 (one-way ANOVA). (C) Measurement of the cell body length of the new-flagellum daughter (NFD) cell and its correlation with the length of the new FAZ for control cells and KIN-G RNAi cells expressing KIN-GT301D. 100 cells were used for measurement for each time point. (D) Effect of the expression of KIN-GT301D on the segregation of flagellar pocket collar (FPC) and basal body (BB). Cells were co-immunostained with anti-TbBILBO1 antibody and YL1/2 antibody. Solid arrowheads and arrows indicate new FPC and new BB, respectively, whereas open arrowheads and arrows indicate old FPC and old BB, respectively. Scale bar: 5 μm. (E, F) Measurement of the inter-FPC distance (E) and inter-BB distance (F) in control cells and KIN-G RNAi cells expressing Thr301 phospho-mimic mutant of KIN-G. 100 cells were used for measurement for each time point, and error bars indicated SD. ****: p<0.0001 (one-way ANOVA).
Figure 7
Phosphorylation of KIN-G at Thr301 by TbPLK disrupts cell division plane placement.
(A) Immunofluorescence microscopy to detect the cell division plane with endogenous triple HA-tagged KLIF in dividing cells from non-induced control and KIN-G RNAi cells. Yellow arrows indicate the KLIF-marked cell division plane. NFD: new-flagellum daughter; OFD: old-flagellum daughter; P: posterior; A: anterior. Scale bar: 5 μm. (B) Immunofluorescence microscopy to detect the new-flagellum daughter (NFD) posterior and the old-flagellum daughter (OFD) nascent posterior with PTP-tagged GB4 protein. Scale bar: 5 μm. (C) Measurement of the inter-posterior distance of bi-nucleated cells from non-induced control and KIN-G RNAi cells. 100 cells were used for measurement for each time point, and error bars indicated SD. ***, p<0.001 (Student’s t-test). (D) Non-induced and tetracycline-induced KIN-G RNAi cells expressing KIN-GT301D. Shown are a non-dividing cell without a visible cleavage furrow and three dividing cells with a visible cleavage furrow. Yellow arrows indicate the cell division plane. Scale bar: 5 μm. (E) Quantitation of bi-nucleated cells with or without a visible cleavage furrow from non-induced and tetracycline-induced KIN-G RNAi cells expressing KIN-GT301D. 100 cells were counted for each time point, and error bars indicated SD from three independent experiments (n=3). ****, p<0.0001 (one-way ANOVA). (F) Percentage of dividing bi-nucleated cells with a normally placed cell division plane or an abnormally placed cell division plane from non-induced and tetracycline-induced KIN-G RNAi cells expressing KIN-GT301D. 100 cells were used for counting for each time point.
Figure 8
Model of KIN-G’s biochemical and cellular function and its regulation through phosphorylation by TbPLK and dephosphorylation by an unknown protein phosphatase.
KIN-G is depicted as a dimer and transports cargos along the microtubule quartet (MtQ) near the centrin arm region to regulate centrin arm biogenesis, which impacts Golgi duplication and flagellum attachment zone (FAZ) elongation, the latter of which promotes flagellum positioning and cell division plane placement, thereby facilitating cytokinesis. The green arrow indicates the direction of movement of KIN-G. Phosphorylation of KIN-G by TbPLK inhibits its microtubule-binding activity, thereby disrupting KIN-G function. Created with BioRender.com.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line | KIN-G RNAi cell line | PMID:39475234; Zhou et al., 2024 | ||
| Other | Serum | MilliporeSigma | F6178 | |
| Chemical compound, drug | Puromycin | MilliporeSigma | P8833 | 1 µg/ml |
| Chemical compound, drug | G418 | Invivogen | ant-gn-5 | 15 µg/ml |
| Chemical compound, drug | Hygromycin | Invivogen | ant-hg-5 | 50 µg/ml |
| Chemical compound, drug | Blasticidin | Invivogen | ant-bl-5b | 10 µg/ml |
| Chemical compound, drug | Phleomycin | Invivogen | ant-ph-5 | 2.5 µg/ml |
| Antibody | Mouse monoclonal anti-HA | MilliporeSigma | H3663; RRID:AB_262051 | 1:400 |
| Antibody | Rabbit polyclonal anti-Protein A | MilliporeSigma | P3775; RRID:AB_261038 | 1:400 |
| Antibody | Mouse monoclonal 20H5 | MilliporeSigma | 04-1624; RRID:AB_10563501 | 1:400 |
| Antibody | Rat monoclonal YL 1/2 | MilliporeSigma | MAB1864; RRID:AB_2210391 | 1:1000 |
| Antibody | Rabbit polyclonal anti-TbPLK | PMID:26272611; Hu et al., 2015 | 1:400 | |
| Antibody | Rabbit polyclonal anti-CC2D | PMID:22114307; Zhou et al., 2011 | 1:1000 | |
| Antibody | Rabbit polyclonal anti-TbCentrin4 | PMID:17567955; Selvapandiyan et al., 2007 | 1:1000 | |
| Antibody | Rabbit polyclonal anti-TbBILBO1 | PMID:18462016; Bonhivers et al., 2008 | 1:400 | |
| Antibody | Rabbit polyclonal anti-TbGRASP | PMID:15138289; He et al., 2004 | 1:400 | |
| Antibody | Rabbit monoclonal anti-ThioP | Thermo Fisher | MA5-32345; RRID:AB_2809626 | 1:5000 |
| Antibody | FITC-conjugated anti-mouse IgG | MilliporeSigma | F5387; RRID:AB_259647 | 1:400 |
| Antibody | FITC-conjugated anti-rabbit IgG | MilliporeSigma | F0382; RRID:AB_259384 | 1:400 |
| Antibody | Cy3-conjugated anti-rabbit IgG | MilliporeSigma | AP187C; RRID:AB_92619 | 1:400 |
| Antibody | Cy3-conjugated anti-rat IgG | MilliporeSigma | AP189C; RRID:AB_92645 | 1:400 |
| Other | VectaShield mounting medium with DAPI | Vector Laboratories | H-1200-10 | |
| Other | EZView anti-HA affinity gel | MilliporeSigma | E6779; RRID:AB_10109562 | |
| Other | Chelating Sepharose Fast Flow | MilliporeSigma | GE17-0575-01 | |
| Chemical compound | GW843682X | Selleck Chemicals | S288001 | |
| Chemical compound | GMP-CPP | Fisher Scientific | NC0641143 | |
| Chemical compound | p-Nitrobenzyl mesylate | MilliporeSigma | SML3810 | |
| Peptide, Recombinant protein | Non-labeled tubulin | Cytoskeleton | T240-A80 | |
| Peptide, Recombinant protein | Rhodamine-labeled tubulin | Cytoskeleton | TL590M | |
| Peptide, Recombinant protein | Lambda protein phosphatase | New England Biolabs | P0753S | 400 units/µl |
| Other | Amicon ultra centrifugal filter, 10 kDa MWCO | MilliporeSigma | UFC801008 | |
| Commercial assay, kit | QuikChange II Site-directed mutagenesis kits | Agilent | 200523 | |
| Software, algorithm | GraphPad Prism | GraphPad Software; https://www.graphpad.com | ||
| Software, algorithm | Photoshop | Adobe; https://www.adobe.com |
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Polo-like kinase phosphorylation of the orphan kinesin KIN-G negatively regulates centrin arm biogenesis in Trypanosoma brucei
eLife 15:RP110793.
https://doi.org/10.7554/eLife.110793.4
