KIN-G is a substrate of TbPLK in T. brucei.

(A). Co-immunostaining of KIN-G-3HA and TbPLK during the cell cycle in the procyclic form. Open arrowheads indicate KIN-G signal at the centrin arm. TbPLK signal at different structures are 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 non-specific 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 12% 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). Phosphosites highlighted in red in the yellow box indicate the in vitro and in vivo phosphosites. (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 S.D. 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 S.D. from three independent experiments.

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. (B). Quantitation of KIN-G-bound microtubules in the presence or absence of TbPLK or in the presence of TbPLKK70R. Error bars indicate S.D. 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 in the presence of TbPLKK70R. Error bars indicate S.D. 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 finally TbPLK was added into the chamber. (E). Quantitation of KIN-G-bound microtubules following the incubation with or without TbPLK. Error bars indicate S.D. from three independent experiments. **: p<0.01; ***: p<0.001 (Student’s t-test).

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. (B). Quantitation of the bound microtubules of KIN-G and its mutants. Error bars indicate S.D. 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 S.D. from three independent experiments. ND: not done. ns: no significance (one-way ANOVA).

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 hours. TbPSA6 serves as a 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 S.D. 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 S.D. from three independent experiments.

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. 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 S.D. ns: no significance; ****: p<0.0001 (Student’s t-test). (C). Effect of the expression of KIN-GT301D on the biogenesis of Golgi and ER 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 S.D. from three independent experiments. ***: p<0.001; ****: p<0.0001 (one-way ANOVA).

TbPLK phosphorylation of Thr301 on KIN-G impairs flagellar inheritance.

(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. 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 S.D. **: p<0.01; ****: p<0.0001 (one-way ANAVA). (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. 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 S.D. ****: p<0.0001 (one-way ANOVA).

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 NFD posterior and the 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 S.D. ***, 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 S.D. 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 measurement for each time point.

Model of KIN-G’s biochemical and cellular function and its regulation by TbPLK phosphorylation.

KIN-G is depicted as a dimer and transports cargos along the MtQ near the centrin arm region to regulate centrin arm biogenesis, which impacts Golgi duplication and 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. The image of microtubules was created in BioRender.com.