Cell Biology

Dynamic localization of the chromosomal passenger complex is controlled by the orphan kinesins KIN-A and KIN-B in the kinetoplastid parasite Trypanosoma brucei

Daniel Ballmer
Bungo Akiyoshi author has email address

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom
The Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, Edinburgh, EH9 3BF, United Kingdom

https://doi.org/10.7554/eLife.93522.2

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Figures and data

KIN-A and KIN-B are bona fide CPC proteins in T. brucei.
(A) Clustered heatmap showing enrichment (log2 intensity based absolute quantification (IBAQ)) of mitotic proteins co-purifying with ectopically expressed GFP-INCENP^CPC1, GFP-KIN-A and GFP-KIN-B. The heatmap was generated using the Python Seaborn library using WPGMA clustering. Cell lines: BAP2190, BAP2286, BAP2288. Immunoprecipitation was performed using anti-GFP antibodies. See Table S2 for all proteins identified by mass spectrometry. (B) Cartoon depicting the kinetoplast (K) / nucleus (N) configuration throughout the cell cycle in procyclic T. brucei, with K* denoting an elongated kinetoplast. The kinetoplast is an organelle found uniquely in kinetoplastids, which contains the mitochondrial DNA and replicates and segregates prior to nuclear division. The KN configuration serves as a cell cycle marker (Siegel et al., 2008; Woodward and Gull, 1990). (C) to (E) Representative fluorescence micrographs showing the dynamic localization of YFP-Aurora B^AUK1 (C), KIN-A-YFP (D) and GFP-KIN-B (E) over the course of the cell cycle. Kinetochores are marked with tdTomato-KKT2. DNA was stained with DAPI. Cell lines: BAP1515, BAP3066, BAP2288. Scale bars, 2 μm. (F) Phylogenetic tree of kinetoplastids, diplonemids and euglenids along with the presence (black dots) / absence (white dots) patterns of CPC components. The phylogenetic tree of Euglenozoa is based on (Butenko et al., 2020).

Kinetochore localization of the CPC depends on KIN-A and KIN-B.
(A) Representative fluorescence micrographs showing the localization of YFP-tagged Aurora B^AUK1, INCENP^CPC1, KIN-A and KIN-B in 2K1N cells upon RNAi-mediated knockdown of indicated CPC subunits. Note that nuclear close-ups are shown here. CPC proteins were not detected in the cytoplasm. RNAi was induced with 1 μg/mL doxycycline for 24 h (KIN-B RNAi) or 16 h (all others). Cell lines: BAP3092, BAP2552, BAP2557, BAP3093, BAP2906, BAP2900, BAP2904, BAP3094, BAP2899, BAP2893, BAP2897, BAP3095, BAP3096, BAP2560, BAP2564, BAP3097. Scale bars, 2 μm. (B) to (E) Quantification of 2K1N cells that have kinetochore-like dots of YFP-tagged Aurora B^AUK1 (B), INCENP^CPC1 (C), KIN-A (D) and KIN-B (E) upon RNAi-mediated depletion of indicated CPC components. All graphs depict the means (bar) ± SD of at least two replicates (shown as dots). A minimum of 100 cells per replicate were quantified. * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (two-sided, unpaired t-test). (F) to (H) Representative fluorescence micrographs showing the localization of ectopically expressed GFP-KIN-A^fl (F), -KIN-A^2-309 (G) and -KIN-A^310-862 (H). Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Arrowheads indicate KIN-A^fl and KIN-A^310-862 signals at kinetochores. KIN-A^2-309 is found at mitotic spindle during metaphase. Cell lines: BAP2286, BAP2297, BAP2287. Scale bars, 2 μm

Structural model of the trypanosome CPC.
(A) Circular view of the BS³ crosslinks observed between the subunits of the trypanosome CPC, obtained from native complexes isolated by immunoprecipitation of YFP-Aurora B^AUK1 (Cell line: BAP2198). pLink2 (Chen et al., 2019) was used to obtain crosslinks from mass spectrometry data. xiView (Graham et al., 2019) was used for data visualization. Only crosslinks with a score better than E^-3 are shown. See Table S2 for all crosslinks identified by mass spectrometry. (B) Cartoon representation showing two orientations of the trypanosome CPC, coloured by protein on the left (Aurora B^AUK1: crimson, INCENP^CPC1: green, CPC2: cyan, KIN-A: magenta, and KIN-B: yellow) or according to their pLDDT values on the right, assembled from AlphaFold2 predictions shown in Figure S3. The pLDDT score is a per-residue estimate of the confidence in the AlphaFold prediction on a scale from 0 – 100. pLDDT > 70 (blue, cyan) indicates a reasonable accuracy of the model, while pLDDT < 50 (red) indicates a low accuracy and often reflects disordered regions of the protein (Jumper et al., 2021). BS³ crosslinks in (B) were mapped onto the model using PyXlinkViewer (blue = distance constraints satisfied, red = distance constraints violated, Cα-Cα Euclidean distance threshold = 30 Å) (Schiffrin et al., 2020).

The CPC is recruited to kinetochores via the KKT7 – KKT8 complex pathway.
(A) Representative fluorescence micrographs showing the localization of YFP-Aurora B^AUK1 upon RNAi-mediated knockdown of KKT7. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell line: BAP577. Scale bars, 2 μm. (B) Quantification of 2K1N cells that have kinetochore-like dots of YFP-Aurora B^AUK1 upon knockdown of KKT7. All graphs depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 50 cells per replicate were quantified. *** P ≤ 0.001 (two-sided, unpaired t-test). (C) Representative fluorescence micrographs showing the localization of YFP-Aurora B^AUK1 upon RNAi-mediated knockdown of KKT9. RNAi was induced with 1 μg/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Cell line: BAP2276. Scale bars, 2 μm. (D) Quantification of 2K1N cells that have kinetochore-like dots of YFP-Aurora B^AUK1 upon knockdown of KKT9. All graphs depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 50 cells per replicate were quantified. *** P ≤ 0.001 (two-sided, unpaired t-test). (E) and (F) Representative micrographs of cells in S phase and anaphase showing recruitment of tdTomato-Aurora B^AUK1 to LacO foci marked by ectopically expressed GFP-KKT7^2-261-LacI (E) or -KKT8-LacI (F). The insets show the magnification of the boxed region. Expression of LacI fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Arrowheads in (F) indicate anaphase kinetochore localization of GFP-KKT8-LacI and tdTomato-Aurora B^AUK1. Anaphase kinetochore localization of tdTomato-Aurora B^AUK1 was observed in 75% of anaphase cells expressing GFP-KKT8-LacI (n = 28). Cell lines: BAP1395, BAP2640. Scale bars, 2 μm. Of note, LacI fusions with INCENP^CPC1, KIN-A and KIN-B constructs robustly localized to kinetochores like their endogenous counterparts and failed to form distinct LacI foci and could therefore not be used to assess ectopic recruitment of KKT proteins. (G) AlphaFold2 model of the KKT7 – KKT8 complex, coloured by protein (KKT7^1-261: green, KKT8: blue, KKT12: pink, KKT9: cyan and KKT11: orange) (left) and by pLDDT (center). BS³ crosslinks in (H) were mapped onto the model using PyXlinkViewer (Schiffrin et al., 2020) (blue = distance constraints satisfied, red = distance constraints violated, Cα-Cα Euclidean distance threshold = 30 Å). Right: Predicted Aligned Error (PAE) plot of model shown on the left (rank_2). The colour indicates AlphaFold’s expected position error (blue = low, red = high) at the residue on the x axis if the predicted and true structures were aligned on the residue on the y axis (Jumper et al., 2021). (H) Circular view of the BS³ crosslinks observed among KKT7 and KKT8 complex subunits, obtained from native complexes isolated by immunoprecipitation of YFP-tagged KKIP1 (Cell line: BAP710a). pLink2 (Chen et al., 2019) was used to obtain crosslinks from mass spectrometry data and xiView (Graham et al., 2019) was used for data visualization. Only crosslinks with a score better than E^-3 are shown. See Table S2 for all crosslinks identified by mass spectrometry. (I) Indicated combinations of 6HIS-tagged KKT8 (∼46 kDa), KKT9 (∼39 kDa), KKT11 (∼29 kDa) and KKT12 (∼23 kDa) were co-expressed in E. coli, followed by metal affinity chromatography and SDS-PAGE. The asterisk indicates a common contaminant.

Two conserved domains within the C-terminal tail of KIN-A promote kinetochore recruitment of the CPC.
(A) Left: AlphaFold2 model of KKT9:KKT11 in complex with KIN-A^700-862. Cartoon representations are coloured by protein (KKT9: cyan, KKT11: orange, KIN-A: magenta) (left) or according to their pLDDT values (blue = high confidence, red = low confidence) (center). Right: Predicted Aligned Error (PAE) plot of model (rank_1) predicted by AlphaFold2 (blue = high confidence, red = low confidence in the relative positions of the domains to one another). CD1 of KIN-A was predicted to interact with KKT9:KKT11 in all five AlphaFold2 models (rank_1 to rank_5). (B) Multiple sequence alignment of KIN-A CD1 and CD2 showing conservation. (C) to (E) Representative fluorescence micrographs showing the localization of tdTomato-Aurora B^AUK1 and YFP-tagged KIN-A^wt (C), KIN-A^ΔCD1 ^(717–743) (D) and KIN-A^ΔCD2 ^(816–862) (E). RNAi was induced with 1 μg/mL doxycycline for 24 h to deplete the untagged KIN-A allele. Cell lines: BAP3067, BAP3128, BAP3127. Scale bars, 2 μm. (F) Stacked bar charts showing the percentage of YFP-tagged KIN-A^wt, KIN-A^ΔCD1 and KIN-A^ΔCD2 on kinetochores, kinetochores + spindle and spindle only in metaphase cells. Examples and schematic illustrations of the three categories used for scoring are presented on the left. A minimum of 50 cells per condition were quantified. (G) Stacked bar charts showing the percentage of tdTomato-Aurora B^AUK1 on kinetochores, kinetochores + spindle and spindle only in metaphase cells upon rescue with YFP-tagged KIN-A^wt, KIN-A^ΔCD1 or KIN-A^ΔCD2. A minimum of 50 cells per condition were quantified. (H) Growth curves for indicated cell lines and conditions. RNAi was induced with 1 μg/mL doxycycline for to deplete the untagged KIN-A allele in the knockdown conditions and cultures were diluted at day 2. Cell lines: BAP3067, BAP3128, BAP3127.

KIN-A ATPase activity is required for central spindle localization of the CPC in anaphase.
(A) Multiple sequence alignment showing conservation of Switch II region in KIN-A and KIN-B, with the key glycine residue (G210 in T. brucei) targeted for rigor mutation highlighted in red. (B) Representative fluorescence micrographs showing the localization of tdTomato-MAP103 (spindle marker) and YFP-tagged KIN-A^wt or KIN-A^G210A (rigor mutant). RNAi was induced with 1 μg/mL doxycycline for 24 h to deplete the untagged KIN-A allele. Cell lines: BAP3068, BAP3071. Scale bars, 2 μm. (C) Quantification showing the percentage of anaphase cells that have YFP-tagged KIN-A^wt or KIN-A^G210A localized at the central spindle. All graphs depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 40 cells per replicate were quantified. *** P ≤ 0.001 (two-sided, unpaired t-test). (D) and (E) Representative fluorescence micrographs showing the localization of tdTomato-Aurora B^AUK1 and YFP-tagged KIN-A^wt (D) or KIN-A^G210A (E). RNAi was induced with 1 μg/mL doxycycline for 24 h to deplete the untagged KIN-A allele. Cell lines: BAP3067, BAP3070. Scale bars, 2 μm. (F) Quantification showing the percentage of anaphase cells that have tdTomato-Aurora B^AUK1 localized at the central spindle upon rescue with YFP-tagged KIN-A^wt or KIN-A^G210A. Graphs for the KIN-A^G210 rescue conditions (grey) depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 30 cells per replicate were quantified. (G) Growth curves for indicated cell lines and conditions. RNAi was induced with 1 μg/mL doxycycline for to deplete the untagged KIN-A allele in the knockdown conditions and cultures were diluted at day 2. Cell lines: BAP3064, BAP3065. (H) Cell cycle profiles for the indicated cell lines and conditions. RNAi was induced with 1 μg/mL doxycycline to deplete the untagged KIN-A allele in the knockdown conditions and cells were fixed after 24 h. All graphs depict the means (bar) ± SD of at least two replicates. A minimum of 300 cells per replicate were quantified. Cell lines: BAP3064, BAP3065. *** P ≤ 0.001 (two-sided, unpaired t-test).

Model for CPC localization and function in trypanosomes.
(A) KIN-A (magenta) and KIN-B (yellow) interact via their coiled-coil domains and form a scaffold for the assembly of CPC2 (cyan) and the catalytic module of the CPC, composed of Aurora B^AUK1 (red) and INCENP^CPC1 (green). During interphase, the catalytic module is positioned close to the kinesin head domains of KIN-A and KIN-B. CPC recruitment to the inner kinetochore is mediated through multiple weak interactions between the C-terminal unstructured tail of KIN-A, containing CD1 and CD2, with the coiled-coil domain of KKT9:KKT11 (dark blue:light blue) and possibly the N-terminus of KKT7 (blue). The KKT8 complex, comprising KKT9:KKT11 and KKT8:KKT12 (dark grey:light grey) subcomplexes, is connected to other kinetochore proteins through KKT7. Additional kinetochore targeting domains of the CPC may reside within the C-terminus of KIN-B and/or CPC2. We propose that the KIN-A:KIN-B subcomplex represents the main localization module of the trypanosome CPC. As illustrated in (B), the affinity of the KIN-A C-terminal tail for its receptor(s) at the kinetochore may be further modulated through phosphorylation by the CDK1 homolog CRK3 and the Aurora B^AUK1 kinase itself (Ballmer et al., 2024). Interaction of the N-terminal motor domain of KIN-A with spindle microtubules (MTs) from prometaphase onwards causes the catalytic module to disengage from its kinesin head-associated state. The ∼100 amino acid long flexible linker within INCENP^CPC1 would then permit Aurora B^AUK1 to phosphorylate its substrates within a larger but nevertheless spatially constrained target area while still being anchored to the kinetochore via KIN-A:KIN-B. The motor domain of KIN-A could thus act as built-in sensor for KT-MT attachments. (C) We propose that the trypanosome CPC is recruited to kinetochores via the KKT7-KKT8 complex pathway (dashed arrow) and that motor activity of KIN-A promotes congression of kinetochores to the metaphase plate during early mitosis. The KKT8 complex dissociates from kinetochores at the metaphase-to-anaphase transition and is possibly degraded in an APC/C-dependend manner. Elimination of the KKT8 complex, the primary kinetochore receptor of the CPC, coupled to MT binding and motor activity of the KIN-A motor domain strips the CPC off kinetochores and facilitates its translocation to the central spindle in anaphase.

KIN-A and KIN-B are bona fide CPC proteins in T. brucei.
(A) Heatmap showing enrichment (log2 intensity based absolute quantification (IBAQ)) of the top 30 proteins, excluding ribosomal proteins, co-purifying with YFP-Aurora B^AUK1. Cell line: BAP73. Immunoprecipitation was performed using anti-GFP antibodies. See Table S2 for all proteins identified by mass spectrometry. (B) and (C) Representative fluorescence micrographs showing the localization of tdTomato-Aurora B^AUK1 and KIN-A-YFP (B) and YFP-KIN-B (C) in late anaphase and telophase cells. Arrows indicate the population of CPC proteins localized to the new FAZ tip. DNA was stained with DAPI. Cell lines: BAP3067, BAP2528. Scale bars, 2 μm.

Depletion of CPC proteins causes growth defects and cell cycle arrest.
(A) Growth curves upon RNAi-mediated knockdown of indicated CPC subunits. Data are presented as the mean ± SD of at least three replicates. RNAi was induced with 1 μg/mL doxycycline and cultures were diluted at day 2. CDS stands for coding sequence, UTR for untranslated region. Cell lines: BAP941, BAP2250, BAP2251, BAP2557, BAP2558, BAP3076. (B) Cell cycle profiles upon knockdown of indicated CPC subunits. RNAi was induced with 1 μg/mL doxycycline and cells were fixed at 16h (for Aurora B^AUK1, INCENP^CPC1 and KIN-A RNAi) or 24 h (CPC2 and KIN-B RNAi). All graphs depict the means (bar) ± SD of at least two replicates. A minimum of 150 cells per replicate were quantified. Cell lines: BAP3092, BAP2554, BAP2557, BAP3093. (C) Western Blot showing levels of YFP-tagged KIN-A or KIN-B upon RNAi-mediated knockdown of KIN-B and KIN-A, respectively. RNAi was induced with 1 μg/mL doxycycline for 16 h (KIN-A) or 24 h (KIN-B). Proteins were detected using anti GFP-antibodies. Asterisk indicates unspecific band. Cell lines: BAP3095, BAP2564. (D) to (F) Representative fluorescence micrographs showing the localization of ectopically expressed GFP-KIN-B^fl (D), -KIN-B^2-316 (E) and -KIN-B^317-624 (F). Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP2288, BAP2289, BAP2290. Scale bars, 2 μm.

AlphaFold2 models of CPC subcomplexes and localization of CPC1 and CPC2 truncations.
(A) to (D) Cartoon representations coloured by protein (left) and pLDDT (center), and PAE plots (right) of AlphaFold2 predictions for indicated CPC subcomplexes. (E) to (J) Representative fluorescence micrographs showing the localization of ectopically expressed GFP-INCENP^CPC1 ^fl (E), - INCENP^CPC1 ^2-147 (F), -INCENP^CPC1 ^2-147 (G), -CPC2^fl (H), -CPC2^2-120 (I) and -CPC2^121-250 (J) in different cell cycle stages. Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP2190, BAP2191, BAP2193, BAP2194, BAP2195, BAP2196. Scale bars, 2 μm.

Kinetochore localization of Aurora B^AUK1 and KIN-A depends on the KKT7 – KKT8 complex pathway.
(A) Representative fluorescence micrographs showing the localization of YFP-Aurora B^AUK1 upon RNA-mediated knockdown of indicated KKT proteins. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP3132, BAP576, BAP2551, BAP2550, BAP3133, BAP1019, BAP2549, BAP2446. Scale bars, 2 μm. (B) Quantification of 2K1N cells that have kinetochore-like dots of YFP-Aurora B^AUK1 upon knockdown of indicated KKT proteins. A minimum of 40 cells per replicate were examined. (C) Representative fluorescence micrographs showing the localization of KIN-A-YFP upon RNA-mediated knockdown of KKT8 complex subunits. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP2970, BAP2968, BAP2969, BAP2971. Scale bars, 2 μm. (D) Quantification of 2K1N cells that have kinetochore-like dots of KIN-A-YFP upon knockdown of indicated KKT8 complex subunits. All graphs depict the means (bar) ± SD of at least two replicates (shown as dots). A minimum of 50 cells per replicate were quantified. (E) Representative micrographs showing recruitment of tdTomato-tagged KKT8, KKT9 and KKT12 to LacO foci marked by ectopically expressed GFP-KKT7^2-261-LacI. Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Cell lines: BAP871, BAP873, BAP874. Scale bars, 2 μm. (F) Representative fluorescence micrograph of an anaphase cell showing localization of tdTomato-KKT8 and ectopically expressed GFP-KIN-A. The insets show the magnification of the boxed region. Expression of GFP-KIN-A was induced with 10 ng/mL doxycycline for 24 h. Cell line: BAP3080. Scale bars, 2 μm. (G) and (H) Cartoon representations coloured by protein (left) and pLDDT (center), and PAE plots (right) of AlphaFold2 predictions for indicated protein complexes. (I) Representative fluorescence micrographs showing localization of YFP-tagged KKT8, KKT12 and KKT9 upon RNAi-mediated knockdown of indicated KKT8 complex subunits. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP2954, BAP2952, BAP2953, BAP2955, BAP2966, BAP2964, BAP2965, BAP2967, BAP2958, BAP2956, BAP2957, BAP2959. Scale bars, 2 μm. (J) to (L) Quantification of 1K*1N and 2K1N cells that have kinetochore-like dots of YFP-tagged KKT8 (J), KKT12 (K) and KKT9 (L) upon knockdown of indicated KKT8 complex subunits. All graphs depict the means (bar) ± SD of at least three technical replicates (shown as dots) from one experiment. A minimum of 100 cells per replicate were quantified. ** P ≤ 0.01, *** P ≤ 0.001 (two-sided, unpaired t-test).

Kinetochore localization of Aurora B^AUK1 and KIN-A depends on the KKT7 – KKT8 complex pathway.
(A) Representative fluorescence micrographs showing the localization of YFP-Aurora B^AUK1 upon RNA-mediated knockdown of indicated KKT proteins. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP3132, BAP576, BAP2551, BAP2550, BAP3133, BAP1019, BAP2549, BAP2446. Scale bars, 2 μm. (B) Quantification of 2K1N cells that have kinetochore-like dots of YFP-Aurora B^AUK1 upon knockdown of indicated KKT proteins. A minimum of 40 cells per replicate were examined. (C) Representative fluorescence micrographs showing the localization of KIN-A-YFP upon RNA-mediated knockdown of KKT8 complex subunits. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP2970, BAP2968, BAP2969, BAP2971. Scale bars, 2 μm. (D) Quantification of 2K1N cells that have kinetochore-like dots of KIN-A-YFP upon knockdown of indicated KKT8 complex subunits. All graphs depict the means (bar) ± SD of at least two replicates (shown as dots). A minimum of 50 cells per replicate were quantified. (E) Representative micrographs showing recruitment of tdTomato-tagged KKT8, KKT9 and KKT12 to LacO foci marked by ectopically expressed GFP-KKT7^2-261-LacI. Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Cell lines: BAP871, BAP873, BAP874. Scale bars, 2 μm. (F) Representative fluorescence micrograph of an anaphase cell showing localization of tdTomato-KKT8 and ectopically expressed GFP-KIN-A. The insets show the magnification of the boxed region. Expression of GFP-KIN-A was induced with 10 ng/mL doxycycline for 24 h. Cell line: BAP3080. Scale bars, 2 μm. (G) and (H) Cartoon representations coloured by protein (left) and pLDDT (center), and PAE plots (right) of AlphaFold2 predictions for indicated protein complexes. (I) Representative fluorescence micrographs showing localization of YFP-tagged KKT8, KKT12 and KKT9 upon RNAi-mediated knockdown of indicated KKT8 complex subunits. RNAi was induced with 1 μg/mL doxycycline for 24 h. Cell lines: BAP2954, BAP2952, BAP2953, BAP2955, BAP2966, BAP2964, BAP2965, BAP2967, BAP2958, BAP2956, BAP2957, BAP2959. Scale bars, 2 μm. (J) to (L) Quantification of 1K*1N and 2K1N cells that have kinetochore-like dots of YFP-tagged KKT8 (J), KKT12 (K) and KKT9 (L) upon knockdown of indicated KKT8 complex subunits. All graphs depict the means (bar) ± SD of at least three technical replicates (shown as dots) from one experiment. A minimum of 100 cells per replicate were quantified. ** P ≤ 0.01, *** P ≤ 0.001 (two-sided, unpaired t-test).

CD1 and CD2 contribute synergistically to kinetochore localization of KIN-A.
(A) Multiple sequence alignment of KIN-A. (B) PAE (left) and pLDDT plots (right) of AlphaFold2 predictions rank1 to rank5 for KIN-A^700-800 in complex with KKT9 and KKT11. (C) to (F) Representative fluorescence micrographs showing the localization of ectopically expressed GFP-KIN-A^310-862 (C), -KIN-A^310-862 ^ΔCD2 (D) and -KIN-B^310-862 ^ΔCD1 (E) and -KIN-A^310-716 (F). Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP2287, BAP2948, BAP2949, BAP2947. Scale bars, 2 μm. (G) Quantification of metaphase (left) and anaphase (right) cells that have kinetochore-like dots of indicated GFP fusion proteins. All graphs depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 150 cells per replicate were quantified. *** P ≤ 0.001 (two-sided, unpaired t-test). (H) Stacked bar charts showing the percentage of KIN-A^ΔCD1-YFP (left) or tdTomato-Aurora B^AUK1 (right) on kinetochores, kinetochores + spindle and spindle in KIN-A^ΔCD1 metaphase cells with and without KIN-A RNAi. A minimum of 70 cells per condition were quantified. Cell line: BAP3128. (I) Cell cycle profiles for the indicated cell lines and conditions. RNAi was induced with 1 μg/mL doxycycline to deplete the untagged KIN-A allele in the knockdown conditions and cells were fixed after 24 h. All graphs depict the means (bar) ± SD of three replicates. A minimum of 500 cells per replicate were quantified. Cell lines: BAP3067, BAP3127, BAP3128.

CD1 and CD2 contribute synergistically to kinetochore localization of KIN-A.
(A) Multiple sequence alignment of KIN-A. (B) PAE (left) and pLDDT plots (right) of AlphaFold2 predictions rank1 to rank5 for KIN-A^700-800 in complex with KKT9 and KKT11. (C) to (F) Representative fluorescence micrographs showing the localization of ectopically expressed GFP-KIN-A^310-862 (C), -KIN-A^310-862 ^ΔCD2 (D) and -KIN-B^310-862 ^ΔCD1 (E) and -KIN-A^310-716 (F). Expression of GFP fusion proteins was induced with 10 ng/mL doxycycline for 24 h. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP2287, BAP2948, BAP2949, BAP2947. Scale bars, 2 μm. (G) Quantification of metaphase (left) and anaphase (right) cells that have kinetochore-like dots of indicated GFP fusion proteins. All graphs depict the means (bar) ± SD of three replicates (shown as dots). A minimum of 150 cells per replicate were quantified. *** P ≤ 0.001 (two-sided, unpaired t-test). (H) Stacked bar charts showing the percentage of KIN-A^ΔCD1-YFP (left) or tdTomato-Aurora B^AUK1 (right) on kinetochores, kinetochores + spindle and spindle in KIN-A^ΔCD1 metaphase cells with and without KIN-A RNAi. A minimum of 70 cells per condition were quantified. Cell line: BAP3128. (I) Cell cycle profiles for the indicated cell lines and conditions. RNAi was induced with 1 μg/mL doxycycline to deplete the untagged KIN-A allele in the knockdown conditions and cells were fixed after 24 h. All graphs depict the means (bar) ± SD of three replicates. A minimum of 500 cells per replicate were quantified. Cell lines: BAP3067, BAP3127, BAP3128.

ATPase activity of KIN-A promotes kinetochore alignment at the metaphase plate.
(A) Multiple sequence alignment of KIN-A and KIN-B from different kinetoplastids, human Kinesin-1, human Mklp2, and yeast Klp9. (B) Microtubule co-sedimentation assay with 6HIS-KIN-A^2-309 (left) and 6HIS-KIN-B^2-316 (right). S and P correspond to supernatant and pellet fractions, respectively. Note that both constructs to some extent sedimented even in the absence of microtubules. Hence, lack of microtubule binding for KIN-B may be due to the unstable non-functional protein used in this study. (C) and (D) Representative fluorescence micrographs showing localization of YFP-tagged KIN-A (C) and KIN-A^G210A (rigor mutant) (D) in G2 and metaphase. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP3066, BAP3069. Scale bars, 2 μm. (E) and (F) Upper: Representative fluorescence micrographs showing localization of YFP-tagged KIN-A (E) and KIN-A^G210A (rigor mutant) (F) in metaphase. The mitotic spindle is marked with tdTomato-MAP103. Bounding boxes indicate area used for generation of intensity profiles in FIJI/Image J. Cell lines: BAP3068, BAP3071. Scale bars, 2 μm. Lower: Example of 1D intensity profiles and corresponding heatmaps from the example cells shown above. (G) and (H) Kinetochore alignment profiles for YFP-tagged KIN-A (G) and KIN-A^G210A (H). RNAi was induced with 1 μg/mL doxycycline for 24 h to deplete the untagged KIN-A allele and cells were treated with 10 μM MG132 for 4 h prior to fixing to prevent anaphase entry (Hayashi and Akiyoshi, 2018). WPGMA clustering was performed using the Python Seaborn library. Cell lines: BAP3064, BAP3065.

ATPase activity of KIN-A promotes kinetochore alignment at the metaphase plate.
(A) Multiple sequence alignment of KIN-A and KIN-B from different kinetoplastids, human Kinesin-1, human Mklp2, and yeast Klp9. (B) Microtubule co-sedimentation assay with 6HIS-KIN-A^2-309 (left) and 6HIS-KIN-B^2-316 (right). S and P correspond to supernatant and pellet fractions, respectively. Note that both constructs to some extent sedimented even in the absence of microtubules. Hence, lack of microtubule binding for KIN-B may be due to the unstable non-functional protein used in this study. (C) and (D) Representative fluorescence micrographs showing localization of YFP-tagged KIN-A (C) and KIN-A^G210A (rigor mutant) (D) in G2 and metaphase. Kinetochores are marked with tdTomato-KKT2. Cell lines: BAP3066, BAP3069. Scale bars, 2 μm. (E) and (F) Upper: Representative fluorescence micrographs showing localization of YFP-tagged KIN-A (E) and KIN-A^G210A (rigor mutant) (F) in metaphase. The mitotic spindle is marked with tdTomato-MAP103. Bounding boxes indicate area used for generation of intensity profiles in FIJI/Image J. Cell lines: BAP3068, BAP3071. Scale bars, 2 μm. Lower: Example of 1D intensity profiles and corresponding heatmaps from the example cells shown above. (G) and (H) Kinetochore alignment profiles for YFP-tagged KIN-A (G) and KIN-A^G210A (H). RNAi was induced with 1 μg/mL doxycycline for 24 h to deplete the untagged KIN-A allele and cells were treated with 10 μM MG132 for 4 h prior to fixing to prevent anaphase entry (Hayashi and Akiyoshi, 2018). WPGMA clustering was performed using the Python Seaborn library. Cell lines: BAP3064, BAP3065.

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