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Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1

Open AccessPublished:December 30, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102853

      ABSTRACT

      The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induce chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

      Keywords

      INTRODUCTION

      Faithful chromosome segregation requires formation of physical linkage between the spindle microtubule (MT) plus ends and the kinetochore (KT), a supramolecular structure composed of ∼ 100 proteins assembled on the chromosomal DNA (
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      ).Though both Ska1 and Dam1 complex can independently bind to the MTs in vitro, their localizations in vivo are mostly confined to the KT-MT junction, but very weakly on the spindle microtubules (
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      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ,
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      • Barnes G.
      Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex.
      ,
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      • Wilson-Kubalek E.
      • Milligan R.A.
      • Grishchuk E.L.
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      Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces.
      ,
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      The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores.
      ), suggesting of other mechanisms that recruit Ska1 to the KTs. Supportively, recent studies indicated that KT localization of Ska in human cells/Dam1 in yeast is regulated by the general MT plus end (+TIP) associated protein, EB1/Bim1 (
      • Thomas G.E.
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      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ,
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      • Klink B.U.
      • Rombaut P.
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      • Janen K.
      • Herzog F.
      • Gatsogiannis C.
      • Westermann S.
      Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.
      ). EB1 is the central regulator of dynamic +TIPs (plus tip tracking proteins) network. It recruits numerous structurally and functionally diverse +TIPs to MT plus ends through direct interaction (
      • Akhmanova A.
      • Steinmetz M.O.
      Tracking the ends: a dynamic protein network controls the fate of microtubule tips.
      ). In human cells, EB1 facilitates chromosome alignment by stabilizing KT localization of the Ska complex (
      • Thomas G.E.
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      • Renjith M.R.
      • Singh P.
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      • Badarudeen B.
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      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ,
      • Thomas G.E.
      • Renjith M.R.
      • Manna T.K.
      Kinetochore-microtubule interactions in chromosome segregation: lessons from yeast and mammalian cells.
      ). However, the mechanism how Ska recruitment to KTs is regulated by EB1 remains to be understood.
      Here, we show that interaction of EB1 to a disordered loop region of Ska1 located in its N-terminus is essential for KT recruitment of Ska complex and metaphase chromosome alignment in human cells. Atomic force microscopy analyses reveal involvement of the Ska1 loop in mediating EB1-Ska1 binding and leading to formation of a complex with defined structure. A conserved motif in the disordered loop of Ska1 is primarily involved in Ska1-EB1 binding and metaphase chromosome alignment. The EB1-binding Ska1 motif bears close similarity with the Serine-any amino acid-Isoleucine-Proline (SXIP) motif of other EB1-binding +TIPs, which mediate their MT recruitment in EB1-dependent manner (
      • Dudziak A.
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      • Herzog F.
      • Gatsogiannis C.
      • Westermann S.
      Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.
      ,
      • Honnappa S.
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      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Akhmanova A.
      • Steinmetz M.O.
      Microtubule +TIPs at a glance.
      ,
      • Busch K.E.
      • Brunner D.
      The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules.
      ,
      • Dzhindzhev N.S.
      • Rogers S.L.
      • Vale R.D.
      • Ohkura H.
      Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends.
      ,
      • Zhang Y.
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      • Liu M.
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      Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration.
      ). Our NMR data further showed that the Ska1 motif-binding affects those residues in EB1, which serve as the binding sites of SXIP motifs of a few distinct +TIPs (
      • Honnappa S.
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      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Jiang K.
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      A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins.
      ,
      • Buey R.M.
      • Sen I.
      • Kortt O.
      • Mohan R.
      • Gfeller D.
      • Veprintsev D.
      • Kretzschmar I.
      • Scheuermann J.
      • Neri D.
      • Zoete V.
      • Michielin O.
      • de Pereda J.M.
      • Akhmanova A.
      • Volkmer R.
      • Steinmetz M.O.
      Sequence determinants of a microtubule tip localization signal (MtLS).
      ). The results demonstrate that Ska stabilization at the KT-MT interface is mediated by Ska1 binding to EB1 through its disordered loop region and primarily, through a conserved motif in the loop. The findings also implicate that formation of KT-MT attachment is facilitated by recognition of EB1 protein by Ska1 at the kinetochore MT plus ends and it involves a mechanism analogous to the EB1-binding +TIPs.

      RESULTS

      Ska1 N-terminal disordered loop is essential for chromosome alignment and Kinetochore localization of Ska1

      Ska1 consists of two structural domains, globular C-terminal domain (residues 133-255), which possesses MT-binding ability (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ) and an N-terminal (1-91) helical domain (Figure 1a) (
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ), which interacts with other components in the Ska complex. These two domains are connected by 40-amino acids disordered loop region, residues (92-132). Deletion of Ska1 loop causes delay in anaphase progression in human cells, though the loop-deleted Ska1 can bind to MTs in vitro similarly as the full-length protein (
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ), suggesting that Ska1 loop has a distinct role during mitosis progression and that is presumably independent of the MT-binding activity of Ska1 mediated by its C-terminus. We therefore aimed to characterize the mitotic defects resulted in the absence of Ska1 loop. This was assessed by expressing a siRNA-resistant Ska1Δloop-GFP construct, in which the loop 92-132 region was deleted (Figure 1b), in HeLa cells under depletion of endogenous Ska1 by siRNA (Figure 1c, d). The results were compared with full length Ska1-GFP expressed cells in parallel. While all the chromosomes in the Ska1-GFP cells could align to the metaphase plate completely, the Ska1Δloop-GFP failed to rescue metaphase alignment of a majority of the chromosomes (Figure 1c). In the Ska1Δ loop-GFP cells, a large subset of chromosomes, though could localize at the spindle midzone, appeared to be aligned only partially to the metaphase plate and the rest appeared to be highly scattered. Under similar condition, the full length Ska1-GFP-expressed cells displayed proper chromosome alignment in majority (∼ 78%) of the cells. Ska1Δloop-GFP expression induced chromosome misalignments in ∼ 88% mitotic cells (Figure 1e). Broadly, three classes of chromosome misalignment defects, Class I, II and III were observed based on the severity of the defects (Experimental Procedures) (Figure S1a) and the sum of the percentages of all three was plotted. Consequently, Ska1Δloop-GFP failed to localize to the KTs (Figures 1f). Same was evident from the intensity plot of KT-localized Ska1-GFP vs. Ska1Δloop-GFP (Figure 1g). The results infer that Ska1 loop is required for chromosome alignment to the metaphase plate and KT localization of Ska1.
      Figure thumbnail gr1
      Figure 1Kinetochore recruitment of Ska1 involves its N terminal disordered loop. (a) Cartoon representation of Ska complex showing the domains of individual Ska proteins (Ska1, Ska2 and Ska3). Ska1, 2, 3 are labeled with different colors. Microtubule-binding C-terminal domains of Ska1 and Ska3 are labeled as MTBD. The N terminal oligomerization domain is labeled as OD. (b) Schematic representation of GFP-tagged full length Ska1 and Ska1 Δ loop constructs. (c) Representative immunofluorescence confocal microscopy images of HeLa cells transfected with control siRNA, Ska1 siRNA, Ska1 siRNA+ siRNA-resistant Ska1-GFP (48 hrs) and Ska1 siRNA+ siRNA- resistant Ska1 Δ loop-GFP (48 hrs). Control and Ska1 siRNA only-treated cells were immunostained with rabbit polyclonal Ska1 antibody (green) and EB1 was stained with rat monoclonal EB1 antibody (red). Ska1-GFP or Ska1 Δ loop-GFP-expressed cells were stained with polyclonal EB1 rabbit antibody. The GFP channels were imaged directly. Microtubules were stained with α-tubulin mouse monoclonal antibody in all cases (violet). DNA was stained with DAPI (shown in white). Scale bar is 5 μm. (d) Western blot images of cell lysates of Ska1 siRNA-, Ska1 siRNA + Ska1-GFP and Ska1 siRNA + Ska1 Δ loop-GFP-treated cells showing expression levels of the exogenous Ska1 proteins with simultaneous depletion of endogenous Ska1. (e) Plot showing percentage of mitotic cells with misaligned chromosomes in Ska1 siRNA-, Ska1 siRNA + Ska1-GFP- and Ska1 siRNA + Ska1 Δ loop-GFP-treated conditions. Data are mean +/- SEM. **** represents P< 0.0001. (f) HeLa cells in Ska1 siRNA + Ska1-GFP or Ska1 siRNA + Ska1 Δ loop-GFP-treated condition were imaged for localization of the GFP fused Ska1 proteins at the kinetochore (KT). Insets 1and 2 represent GFP tagged Ska1 proteins and CENP-A, respectively. EB1 was stained with rabbit EB1 antibody, and CENP-A was stained with mouse monoclonal CENP-A antibody. DNA was stained with DAPI. The scale bars in the main and inset figures are 5 μm and 1 μm, respectively. (g) Plot showing the intensity of Ska1-GFP vs. Ska1 Δ loop-GFP at individual KTs in HeLa cells. Data are mean +/- SEM. **** P< 0.0001 (n = 3). ∼ 100 KTs in each of the three experiments were measured.

      Ska1 interacts with EB1 through the loop and its KT recruitment requires EB1

      EB1 is essential for KT localization of Ska1 and it interacts with Ska1 (
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ). We therefore asked whether the Ska1 loop is involved in Ska1-EB1 interaction, which in turn can promote KT recruitment of Ska1. We first examined the role of Ska1 loop in Ska1-EB1 binding. While the IP of endogenous EB1 in HEK 293T cells showed presence of the full length Ska1-GFP, the Ska1Δloop-GFP did not show its detectable presence in the IP (Figure 2a). Similarly, reverse pulldown of Ska1Δloop-GFP by GFP trap did not show any presence of endogenous EB1 in the lysates of Ska1Δloop-GFP expressed cells under endogenous Ska1 knockdown by Ska1 siRNA; though under similar condition, the pulldown of full length Ska1-GFP showed strong presence of EB1 (Figure 2b). Therefore, Ska1 loop is essential for Ska1-EB1 binding.
      Figure thumbnail gr2
      Figure 2Ska1 interacts with EB1 through its loop and its KT localization requires EB1. a) Ska1-GFP and Ska1 Δloop-GFP transfected HEK 293T cells were mitotic synchronized by double thymidine and subjected to immunoprecipitation (IP) by using EB1 antibody and the samples were analyzed for the presence of the Ska1 proteins by Western blotting. b) Double thymidine synchronized mitotic lysates of Ska1-GFP-and Ska1 Δloop-GFP-transfected HEK293T cells treated with Ska1 siRNA were immunoprecipitated using GFP trap beads followed by Western blotting to probe the presence of EB1. c) GFP tagged Ska1 1-132 and Ska1 1-91 expressed HEK293T cells were synchronized and the mitotic cell lysate was subjected to immunoprecipitation by using GFP trap beads. The presence of EB1 and GFP-tagged Ska1 proteins were detected by Western blotting. Rabbit or mouse IgG, wherever applicable, was used as control in all these experiments. (d) Representative immunofluorescence images of inducible EB1 knockout HeLa cells that were transfected with Ska1 siRNA and Myc-Ska1 1-132. Insets 1 and 2 represent Myc-Ska1 1-132 levels at the kinetochores in control and EB1 knockout cells, respectively. The scale bars in main and inset figures are 5 μm and 1 μm, respectively. EB1 knockout (EB1 KO) was induced by treating cells with doxycycline for four days. Control (without doxycycline) and EB1 KO cells were stained with Myc monoclonal antibody and polyclonal antibody against ACA. DNA was stained with DAPI. (e) Intensity of Myc-Ska1 1-132 localized at individual KT in control vs. EB1 KO cells. ∼ 500 KTs from three different experiments were counted in each case. (f) Plot showing percentage of mitotic cells with misaligned chromosomes in Ska1 siRNA + Myc Ska1 1-132 in control vs. EB1 knockout condition. (g) Cartoon representations of Ska1 1-132-GFP, Ska1 1-91-GFP and Myc Ska1 1-132 constructs.
      Ska1 (1-132), consisting of the KT-targeting structural domain (1-91) followed by the loop, can localize to KTs (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ,
      • Sivakumar S.
      • Janczyk P.L.
      • Qu Q.
      • Brautigam C.A.
      • Stukenberg P.T.
      • Yu H.
      • Gorbsky G.J.
      The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores.
      ,
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ), whereas we found that the structural domain of Ska1 (1-91) fails to localize to KTs in HeLa cells (Figure S1b). It was also observed that Ska1 (1-132), but not Ska1 (1-91), interacts with EB1 (Figure 2c). Though the amount of EB1 associated with Ska1 1-132-GFP as revealed by GFP nanobody conjugated bead (GFP trap) method showed relatively less presence of EB1 as compared to EB1 associated with the full length Ska1-GFP, possibly due to some interference caused by the GFP nanobody-fused bead (compare Figure 2c and Figure 2b), the IP of Ska1 1-132-GFP by using GFP antibody showed a strong presence of EB1 (Figure S1c). A reverse IP using EB1 antibody also showed strong presence of Ska1 1-132 GFP (Figure S1d). Since the loop is essential for EB1 binding, we then checked whether KT recruitment of Ska1 1-132, which contains the loop, requires EB1. Myc-Ska1 1-132 was expressed in CRISPR-Cas9 based EB1 conditional knockout (EB1 KO) HeLa cells under doxycycline treatment (Experimental Procedures) and its KT localization was assessed as compared to the control HeLa cells without doxycycline. As expected, the doxycycline-treated cells showed robust loss of EB1 expression (
      • McKinley K.L.
      • Cheeseman I.M.
      Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects.
      ) (Figure S1e-f). KT localization of Myc-Ska1 1-132 was substantially impaired in the EB1 KO cells as compared to control (Figure 2d). The insets show better visualization of the differences of KT localized Myc- Ska1 1-132 (Insets Figure 2d). Intensity analysis of individual KTs showed significantly reduced level of Myc-Ska1-1-132 in the EB1 KO cells (Figure 2e). To rule out the possibility of any influence of altered microtubule dynamics due to EB1 knockout, if any, on KT localization of Ska1 , we imaged HeLa cells expressed with Myc Ska1 1-132 in the presence of 300 nM nocodazole, which affects microtubule dynamics (
      • Vasquez R.J.
      • Howell B.
      • Yvon A.M.
      • Wadsworth P.
      • Cassimeris L.
      Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.
      ). KT localization of Myc Ska1 1-132 appeared to be minimally affected in the nocodazole-treated cells compared to its absence (Figure S1g-h). Consequently, chromosome misalignment defects were induced in the Myc-Ska1 1-132 expressed EB1 KO cells. ∼88% mitotic cells had misaligned chromosomes in the Myc Ska1 1-132-expressed EB1 KO cells; whereas only 29% Myc Ska1 1-132-expressed control cells showed the misalignment defects (Figure 2f). Together, the results indicate that binding of Ska1 loop to EB1 is essential for KT recruitment of Ska1.

      Conserved motif in Ska1 loop is critical for EB1-Ska1 interaction and metaphase chromosome alignment.

      Several +TIPs are recruited to MT plus ends by binding to EB1 through their conserved SXIP motif. Mutation of the hydrophobic IP of SXIP motif to NN (SHNN) disrupts EB1 binding and their MT plus end recruitment (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ), emphasizing crucial role of the hydrophobic moiety provided by the residues I and P of SXIP for their EB1-dependent plus end recruitment. Human Ska1 consists of a similar motif with sequence SHLP in the upstream region in its loop and the motif is conserved in several vertebrates (Figure 3a). We investigated possible role of this motif in EB1 binding by mutating LP of the motif to NN (Figure 3b) (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ). Pull-down of wild-type Ska1-GFP in HEK-293 cells depleted of endogenous Ska1 showed presence of EB1, but not in the pull-down Ska1-SHNN-GFP, supporting essential role of Ska1 SHLP motif for Ska1-EB1 interaction (Figure 3c). This conclusion was further strengthened by GST pull-down using GST-tagged EB1, which could efficiently pulldown purified recombinant 6x His tagged wild type Ska1, but not 6xHis Ska1 SHNN or Ska1ΔSHLP (the whole motif deleted version). Thus, wild-type Ska1 could associate with EB1-GST strongly, but the same was impaired drastically both in the case of Ska1 SHNN mutant and Ska1 ΔSHLP (Figure 3d). As expected, 6xHis Ska1 Δloop also did not show any association with EB1 GST. Previous studies developed a SXIP peptide aptamer (ALNGQSRIPVLKRHTR) that binds to EB1 strongly and interferes with plus end targeting of several EB1-binding proteins (
      • Lesniewska K.
      • Warbrick E.
      • Ohkura H.
      Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics.
      ,
      • Ayyappan S.
      • Dharan P.S.
      • Krishnan A.
      • Marira R.R.
      • Lambert M.
      • Manna T.K.
      • Vijayan V.
      SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer.
      ). Therefore, we also checked if the EB1-binding SXIP peptide aptamer interferes with Ska1-EB1 interaction. GST pull-down assay showed that the peptide aptamer inhibits 6xHis-Ska1 binding to EB1-GST in a dose dependent manner. At 1: 5 molar ratio of EB1: peptide, EB1-Ska1 association was reduced by ∼ 70% (Figure S2a).
      Figure thumbnail gr3
      Figure 3SHLP motif of Ska1 is essential for its kinetochore localization and chromosome alignment in cells. (a) Amino acid sequences of SHLP-motif containing Ska1 region of humans (hs), Pan troglodytes (pt), Macaca mulatta (mm), Canis lupus familiaris (clf), Bos taurus (bt), Mus musculus (m. mus), Rattus norvegicus (rn), Danio rerio (dr), and Xenopus tropicalis (xt). Dotted region represents the SHLP or SHLP-like motifs in the proteins. The bars represent the conservation scores of the amino acids in the species. Scale 1 to 10 (represented as *). (b) Schematic representations of EB1-GST, wild type Ska1 and various Ska1 mutant constructs. (c) Double thymidine synchronized mitotic cell lysates of Ska1-GFP-and Ska1 SHNN-GFP-transfected HEK 293T cells were immunoprecipitated using GFP trap beads followed by Western blotting to probe EB1. (d) Mixture of recombinant 6xHis tagged Ska1 or Ska1 Δ loop, or Ska1 ΔSHLP or Ska1 SHNN with EB1-GST was subjected to GST pull-down and the association of EB1-GST with Ska1 WT vs. Ska1 mutant proteins was probed by Western blot analysis of the pull-down samples. (e) Representative confocal images of Ska1 siRNA-transfected HeLa cells were expressed with Ska1-GFP or Ska1 SHNN-GFP for 48 hrs prior to staining with EB1 rabbit antibody and Hec1 mouse antibody. GFP-tagged proteins were imaged directly. DNA was stained with DAPI. (f) Plot showing the percentage of mitotic cells with chromosome alignment defects in the Ska1 WT vs. Ska1 SHNN-GFP-expressed cells. ∼60-80 mitotic cells counted in each experiment (no of experiments = 3 for each). Data are mean +/- SEM. (g) Plot shows the intensity of KT localized Ska1-GFP and Ska1 SHNN-GFP normalized to Hec1 in Ska1-GFP and Ska1 SHNN-GFP expressed cells, respectively. ∼ 300 KTs from three experiments were analyzed in each case. (h) Ska1 or Ska1 ΔSHLP (0.5 μM) was added to the pre-polymerized MTs in vitro in the presence or absence of EB1 (1 μM) and then stained with antibodies against α-tubulin, Ska1 and EB1. (i) Intensity plot of Ska1/ Ska1 ΔSHLP localized onto MTs in the presence and absence of EB1. Number of MTs analyzed ∼ 120 in each case. **** refers to P<0.0001, * P<0.05 (n = 3). Scale bar in all images = 5 μm.
      Next, we examined if Ska1 SHLP to SHNN mutation affects KT recruitment of Ska1. Expression of Ska1-SHNN-GFP under depletion of endogenous Ska1 (Figure S2c) resulted in metaphase chromosome misalignments in ∼ 60% mitotic cells (Figure 3e, f). KT localization of Ska1 was also substantially reduced in the Ska1-SHNN-GFP-expressed cells (Figure 3g). Similar defects were observed in cells expressed with the SHLP-deleted Ska1 variant (Ska1-ΔSHLP-GFP) (Figure S2b). We also found that SHNN mutation does not affect Ska1 binding to Ska3 since the GFP pulldown from Ska1-GFP or Ska1-SHNN-GFP-expressed cells show Ska3 association to similar extent, suggesting that Ska complex assembly is not affected by SHNN mutation (Figure S2d). However, KT localization of Ska3 was also reduced significantly in the Ska-SHNN-GFP expressed cells as compared to Ska1-GFP control cells (Figure S2e, f), indicating that KT localization of not Ska1 alone, but of the whole Ska complex is impaired in the SHNN mutant condition. We next sought to determine the effect of the absence of SHLP motif on Ska1 localization on MTs. Purified 6xHis WT Ska1 or the SHLP motif-deleted Ska1 (Ska1 ΔSHLP) was added to pre-polymerized MTs in vitro in the presence of EB1 and Ska1 localization on the MTs was assessed by fluorescence microscopy. WT Ska1 was localized on the MTs to a significantly greater extent as compared to Ska1 ΔSHLP (about 1.6 folds) (Figure 3h, i), supporting that EB1 interaction with Ska1 SHLP motif facilitates Ska1 recruitment onto MTs.

      High-speed AFM imaging shows association of Ska1 with EB1 dimer through its loop

      To visualize the interacting domains of Ska1 and EB1 during their complex formation in high resolution in real time, the dynamics of recombinant EB1, Ska1 and their mixture were imaged by high-speed AFM (HS-AFM). This laboratory-built AFM integrated with a high-speed recording device (
      • Ando T.
      • Uchihashi T.
      • Kodera N.
      High-speed AFM and applications to biomolecular systems.
      ,
      • Davies T.
      • Kodera N.
      • Kaminski Schierle G.S.
      • Rees E.
      • Erdelyi M.
      • Kaminski C.F.
      • Ando T.
      • Mishima M.
      CYK4 promotes antiparallel microtubule bundling by optimizing MKLP1 neck conformation.
      ,
      • Kodera N.
      • Noshiro D.
      • Dora S.K.
      • Mori T.
      • Habchi J.
      • Blocquel D.
      • Gruet A.
      • Dosnon M.
      • Salladini E.
      • Bignon C.
      • Fujioka Y.
      • Oda T.
      • Noda N.N.
      • Sato M.
      • Lotti M.
      • Mizuguchi M.
      • Longhi S.
      • Ando T.
      Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.
      ) allowed capturing the domain organization and dynamics of the proteins at single molecule level within a time scale as fast as 150 milli-second per frame. Protein solutions were drop casted onto mica surface and the movements of the proteins were captured. EB1 protein alone was found to be majorly in the dimer form with its two N-terminal globular calponin homology (CH) domains (referred as EB1-N), separated from each other and a relatively flat and extended bar-like structure (referred as EB1-C), likely the EB1 C-terminal dimer (Figure 4a). Rapid dynamics of the EB1 N and C domains was observed (Movie S1). Identity of EB1-N and EB1-C domains was confirmed from their average heights in the AFM images (Figure 4d-e). The Gaussian plots of height of EB1-N and EB1-C vs. the number of frames with the corresponding heights are shown in Figure 4d-e. The maximum height that was displayed in majority of the frames was considered as the average height of the respective domain. The heights of EB1 N and EB1 C as determined from the AFM data were consistent with their crystal structures (Figure S3d-e) (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Buey R.M.
      • Sen I.
      • Kortt O.
      • Mohan R.
      • Gfeller D.
      • Veprintsev D.
      • Kretzschmar I.
      • Scheuermann J.
      • Neri D.
      • Zoete V.
      • Michielin O.
      • de Pereda J.M.
      • Akhmanova A.
      • Volkmer R.
      • Steinmetz M.O.
      Sequence determinants of a microtubule tip localization signal (MtLS).
      ,
      • Buey R.M.
      • Mohan R.
      • Leslie K.
      • Walzthoeni T.
      • Missimer J.H.
      • Menzel A.
      • Bjelic S.
      • Bargsten K.
      • Grigoriev I.
      • Smal I.
      • Meijering E.
      • Aebersold R.
      • Akhmanova A.
      • Steinmetz M.O.
      Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice.
      ,
      • Honnappa S.
      • Okhrimenko O.
      • Jaussi R.
      • Jawhari H.
      • Jelesarov I.
      • Winkler F.K.
      • Steinmetz M.O.
      Key interaction modes of dynamic +TIP networks.
      ,
      • De Groot C.O.
      • Jelesarov I.
      • Damberger F.F.
      • Bjelic S.
      • Scharer M.A.
      • Bhavesh N.S.
      • Grigoriev I.
      • Buey R.M.
      • Wuthrich K.
      • Capitani G.
      • Akhmanova A.
      • Steinmetz M.O.
      Molecular insights into mammalian end-binding protein heterodimerization.
      ). The AFM images additionally revealed the structural organization of the linker region connecting two EB1 N domains and EB1 C and it appeared as a flexible thin linker. Notably, a rapid length change of ranging from ∼ 0 to 24 nm and concomitant stretching of the N-domains from the C-terminal dimer domain were evident (Movie S1). Majority (∼70%) of the frames displayed an average stretching length between ∼ 4 to 12 nm (Figure S3b). HS-AFM imaging of Ska1 revealed that it exists exclusively in the monomer form, with its large C-terminal globular domain, a slightly extended N-terminus and the loop region connecting the two structural domains (Figure 4b). Ska1 structural domains also exhibited dynamic stretching from each other, likely due to the connecting loop region (Movie S2). Although the two structural domains showed a minimum 2 nm and maximum 16 nm stretching, about 70% of the frames showed an average oscillation between 6 to 13 nm distance (Figure S3c). The individual domains of Ska1 (Ska1 N and Ska1 C) were identified based on their maximum height analysis in the AFM images (Figure 4f-g). Ska1 C domain was identified by cross-verifying the average length from its crystal structure of Ska1C (
      • Jeyaprakash A.A.
      • Santamaria A.
      • Jayachandran U.
      • Chan Y.W.
      • Benda C.
      • Nigg E.A.
      • Conti E.
      Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface.
      ,
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ) with the maximum heights measured from the AFM images (Figure S3f). Images of the mixture of EB1 and Ska1 showed formation of slightly curved extended structures with average length ∼30-35 nm (Figure 4c, Figure S3a). Ska1 appeared to be anchored to the bar-like coiled-coil EB1 C dimer structure through its flexible loop, while the globular Ska1 C region moves around the globular EB1 N domains and then positions itself vertically resulting in an extended structure with a slight curvature (Figure 4c, Movie S3). Several such molecules with the similar structural organization were observed (Figure S3a).
      Figure thumbnail gr4
      Figure 4Molecular dynamics of EB1, Ska1 and EB1-Ska1 complex. HS-AFM images and schematic representations of EB1, Ska1 and EB1-Ska1 complex. a) HS-AFM images showing the dynamic changes of the domains of EB1 dimer (10 nM), b) Ska1 monomer (10 nM) and c) the same in the EB1-Ska1 complex (both 10 nM). The representative frames as shown are taken from the time lapse images (corresponding movies provided in ) of EB1, Ska1 and EB1- Ska1 complex, respectively. Images were captured at 0.15 seconds per frame of 50x50 nm2 size at 80 x 80 pixel. The colour scale was set at 4.25 nm for all the frames. Scale bar = 10 nm. The schematics of different domains of EB1 and Ska1 were drawn freehand to represent the corresponding HS-AFM images. The arrowheads of different colours as shown denote the domains and loops of EB1 and Ska1 as per the schematics drawn. (d-g) The plots represent the height distributions of EB1 N, EB1 C, Ska1 N and Ska1 C domains with respect to number of frames. The lines in the plots are based on best fitting of the data.

      Ska1 motif binds to residues in EB1 that are targeting sites of +TIPs.

      As SHLP motif in Ska1 loop is critical for EB1-mediated Ska1 recruitment to KTs and Ska1-EB1 interaction, we sought to identify the residues in EB1 that make contacts with the SHLP motif region of Ska1 by NMR. We studied the interaction between EB1 and a synthesized Ska1 peptide, Ska1 p with amino acid sequence KENVPSHLPQVTVT, which consists of the SHLP motif and its flanking region in both sides spanning from residues 88-101. Peptide was synthesized using solid phase peptide synthesizer and purified using High-Performance Liquid Chromatography (HPLC). Intense base peak at m/z value ∼1590 corresponding to the molecular weight of the peptide is shown in Figure S4b. To probe the interaction between the Ska1 SHLP peptide, Ska1 p and EB1, 15N-1H TROSY of 2H-15N labelled EB1 was measured with increasing concentrations of Ska1 p. After each addition of the peptide, pH was adjusted to 6.8. The overlaid spectra of EB1 in the presence and absence of Ska1 p showed a significant dose-dependent chemical shift changes of specific amino acids located within the EBH domain (residues 210-260 of human EB1) (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ) of EB1 upon addition of Ska1 p (Figures 5a-f, S4a).
      The residues 248A, 255I, 249T, 252G, 251E, 233G and 232E in the EBH domain of EB1 showed significant change in their chemical shift values, when bound to the Ska1 SHLP peptide (Figure 5 a-f, and S4a). The chemical shift changes of all the amino acids that were affected by the Ska1 SHLP peptide binding is shown in Figure 4g. It is interesting to note that the amino acids affected by Ska1 p binding largely overlap with the binding sites of other +TIPs proteins including MACF, APC (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ). The amino acids showing significant change in their chemical shifts upon binding to the Ska1 p were also mapped on the X-ray crystal structure of EB1 C-terminus (
      • Honnappa S.
      • Okhrimenko O.
      • Jaussi R.
      • Jawhari H.
      • Jelesarov I.
      • Winkler F.K.
      • Steinmetz M.O.
      Key interaction modes of dynamic +TIP networks.
      ) and compared with those of the SXIP peptide aptamer/Ska1 SHLP peptide bound to EB1 (Figure S4c-d). Interestingly, the residues affected by Ska1 SHLP peptide are nearly the same subset of amino acids that showed large chemical shift deviations upon binding of EB1 to the SXIP aptamer peptide (
      • Ayyappan S.
      • Dharan P.S.
      • Krishnan A.
      • Marira R.R.
      • Lambert M.
      • Manna T.K.
      • Vijayan V.
      SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer.
      ) and they are conserved across several metazoan species (Figure 4h). Furthermore, nearly the same subset of amino acids was previously shown by NMR to be involved in binding of EB1 with the SXIP motif of +TIP, MACF (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ) (Figure S4e). These results together demonstrate that Ska1 SHLP motif binds to unique sites in EB1 that are specific for the SXIP -type +TIPs.
      Figure thumbnail gr5
      Figure 5Ska1 SHLP peptide binds to amino acids in EB1 C-terminus. Overlaid 15N-1H TROSY spectra of EB1 alone and in the Ska1 SHLP peptide (Ska1 p)-bound states. (a-b) TROSY spectra 15N-1H labelled EB1 (95 μM) only; 15N-1H EB1 (95 μM) plus Ska1 p (19 μM); 15N-1H EB1 (95 μM) plus Ska1 p (66.5 μM) are shown. A number of residues in the C-terminal region of EB1 showed significant changes in their chemical shift values. (c-f) Close-up views of the representative cross peaks with the large chemical shift changes of the amino acids, 248A, 232E, 249T and 252G of EB1 are shown. (g) Chemical shift perturbations of the amino acids in EB1 based on the 15N-1H TROSY spectra of EB1 upon addition of Ska1 p (66.5 μM) are shown. (h) Conservation of the amino acids of EB1 from different species that were affected significantly upon Ska1 SHLP peptide binding.

      DISCUSSION

      Ska1 plays critical role in the formation of stable KT-MT end-on attachment and sister kinetochore biorientation. It stabilizes Ska complex association with the outer KT by binding with Ska3 and also by mediating interaction with NDC80 primarily through its N-terminal coiled-coil domain (1-91) (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ,
      • Jeyaprakash A.A.
      • Santamaria A.
      • Jayachandran U.
      • Chan Y.W.
      • Benda C.
      • Nigg E.A.
      • Conti E.
      Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface.
      ,
      • Auckland P.
      • Clarke N.I.
      • Royle S.J.
      • McAinsh A.D.
      Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment.
      ,
      • Huis In 't Veld P.J.
      • Volkov V.A.
      • Stender I.D.
      • Musacchio A.
      • Dogterom M.
      Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling.
      ,
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ). However, Ska1 (1-91) alone fails to localize to KTs in cells, suggesting that MT binding of Ska1 is a prior event, which then allows the complex to establish connection with the outer kinetochore. Though Ska1 has an intrinsic MT-binding site in its C-terminal globular domain (133-255), Ska1 binding to purified MTs induces destabilization of MTs by facilitating more curvature to the depolymerizing MTs (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ). Additionally, previous studies and our data here (Figure 2d) have shown that Ska1 without its MT-binding C-terminal domain can still be recruited to the KTs efficiently (
      • Sivakumar S.
      • Janczyk P.L.
      • Qu Q.
      • Brautigam C.A.
      • Stukenberg P.T.
      • Yu H.
      • Gorbsky G.J.
      The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores.
      ,
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ,
      • Sivakumar S.
      • Gorbsky G.J.
      Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska) complex to kinetochores.
      ). All these observations indicate that less likely the intrinsic MT-binding region of Ska1, but additional molecular interactions involving other regulatory site of Ska1 could be attributed to Ska1/Ska complex stabilization at the MT-KT interface in vivo. Supportively, we have shown here that interaction between the N-terminal loop (92-132) of Ska1 with MT plus end-associated protein EB1 is required for Ska1 stabilization at the KTs and metaphase chromosome alignment (Figure 1). Previous study by time-lapse imaging showed that absence of the Ska1 loop causes significant delay in anaphase progression and chromosome alignment defects specifically at the stage just prior to anaphase entry (
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ). The chromosomes appeared to be loosely congressed as they spread over on a wider region on both sides of the metaphase plate. Consistently, our data of fixed cell images of the metaphase arrested cells also showed a similar chromosome organization defect in the absence of the Ska1 loop. Additionally, scattered organization of some chromosomes near the spindle pole area was also observed (Figure 1c). Interestingly, our results also revealed that a conserved motif, SHLP in the Ska1 loop region is primarily involved in regulating interaction of Ska1 with EB1 (Figure 3). Abrogation of interaction specific to the motif is sufficient to result in the chromosome alignment defects analogous to the functional abrogation of not only the Ska1 protein alone, but of the whole Ska complex at the KT, since we showed that Ska1 SHLP motif mutation disrupts KT localization of both Ska1 and Ska3 (Figure 3, S2). This functional impairment is not due to any defects in Ska complex assembly per se, since Ska1 binding to Ska3 was not affected upon mutation of the motif (Figure S2). Previous studies showed that Ska complex localization to kinetochore requires interaction of Ndc80 with Ska3 in a Cdk1-mediated Ska3 phosphorylation-dependent manner (
      • Huis In 't Veld P.J.
      • Volkov V.A.
      • Stender I.D.
      • Musacchio A.
      • Dogterom M.
      Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling.
      ,
      • Zhang Q.
      • Sivakumar S.
      • Chen Y.
      • Gao H.
      • Yang L.
      • Yuan Z.
      • Yu H.
      • Liu H.
      Ska3 Phosphorylated by Cdk1 Binds Ndc80 and Recruits Ska to Kinetochores to Promote Mitotic Progression.
      ). Here, our results indicate that the interaction of Ska1 with EB1 is essential for the kinetochore recruitment of Ska complex. It is possible that both mechanisms are involved in this process. Since LP to NN mutation in Ska1 loop impairs both EB1 binding and KT localization of Ska1 (Figure 3), and this mutation site is outside the Ska3-binding region, a likely possibility is that Ska1 interaction with EB1could facilitate Ska3 interaction to Ndc80/kinetochore. The idea that EB1-Ska complex interaction on the microtubules occurs prior to Ska interaction with the kinetochore is also supported by the fact that Cdk1 sites-specific Ska3 phospho-deficient mutant can localize to the microtubules but not to the kinetochore (
      • Zhang Q.
      • Sivakumar S.
      • Chen Y.
      • Gao H.
      • Yang L.
      • Yuan Z.
      • Yu H.
      • Liu H.
      Ska3 Phosphorylated by Cdk1 Binds Ndc80 and Recruits Ska to Kinetochores to Promote Mitotic Progression.
      ). As the sequence of the Ska1 motif bears close similarity with the general EB1-binding SXIP motif of several MT plus end-targeting +TIPs and the SHLP motif binds to residues in EB1 that the SXIP motifs of +TIPs bind to, it is reasonable to think that Ska1 is targeted to the kinetochore-targeting MT plus ends in vivo in a similar way that other EB1-binding +TIPs do. This mechanism could be conserved across eukaryotes since the functional homolog of Ska in yeast, the Dam1 complex has also been shown to interact with yeast EB1, Bim1 through a similar motif (
      • Dudziak A.
      • Engelhard L.
      • Bourque C.
      • Klink B.U.
      • Rombaut P.
      • Kornakov N.
      • Janen K.
      • Herzog F.
      • Gatsogiannis C.
      • Westermann S.
      Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.
      ).
      Our AFM data enabled to visualize the dynamics of organization of different regions of EB1 and Ska1 proteins both individually and together during their complex formation. They also enabled to identify the structural regions of the two proteins that mediate interaction during their complex formation. The Ska1 molecule appeared to associate with the C-terminal dimer region of EB1 with its N-terminal flexible loop region during formation of the Ska1-EB1 complex (Figure 4). While the loop being anchored, the structural domains of both the proteins reorganize in a manner that leads to formation of a slightly curved extended structure. Although the functional relevance of such unique structural organization is not clear at this time, it is likely that in this configuration, the MT binding domains of the two proteins (two EB1 N calponin homology domains and one Ska1 C domain) are positioned towards one side. Such arrangement could favour increased MT binding of Ska1. Supportively, our data showed increased Ska1 localization onto purified MTs, when added together with EB1 (Figure 3g). Majority of the Ska1-EB1 structures appeared to have slight curvature. We had shown previously that Ska1-EB1 complexes form distinct MT-bound structures in vitro and the structures were also curved in nature as they wrap around the MT lattice (
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ). However, unlike the EB1-Ska1 complex structures in the absence of MTs shown here, the MT-bound structures appeared more extended and they decorated nearly the whole MT lattice longitudinally (
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ). It is possible that the MT surface facilitates formation of such extended structures by localizing many Ska1-EB1complexes closely on the MT surface.
      The SXIP docking sites of nearly all the SXIP +TIPs lie within the EB homology (EBH) domain of EB1 C-terminus (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Ayyappan S.
      • Dharan P.S.
      • Krishnan A.
      • Marira R.R.
      • Lambert M.
      • Manna T.K.
      • Vijayan V.
      SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer.
      ,
      • Gireesh K.K.
      • Shine A.
      • Lakshmi R.B.
      • Vijayan V.
      • Manna T.K.
      GTP-binding facilitates EB1 recruitment onto microtubules by relieving its auto-inhibition.
      ). Usually, the apolar I and P residues of SXIP are involved in packing interaction with the hydrophobic cleft of EBH and mutation of these sites to polar residues, such as Asparagine largely interferes with EB1 binding (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Buey R.M.
      • Sen I.
      • Kortt O.
      • Mohan R.
      • Gfeller D.
      • Veprintsev D.
      • Kretzschmar I.
      • Scheuermann J.
      • Neri D.
      • Zoete V.
      • Michielin O.
      • de Pereda J.M.
      • Akhmanova A.
      • Volkmer R.
      • Steinmetz M.O.
      Sequence determinants of a microtubule tip localization signal (MtLS).
      ). We showed that mutation of LP to NN in Ska1 SHLP abrogates Ska1-EB1 interaction and it leads to chromosome alignment defects (Figure 3). Furthermore, Ska1 SHLP motif binds to the EBH domain and the set of residues in EBH affected by Ska1 SHLP binding majorly overlaps with those involved in other key SXIP-type +TIPs binding (Figure 5, Figure S4) (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Buey R.M.
      • Sen I.
      • Kortt O.
      • Mohan R.
      • Gfeller D.
      • Veprintsev D.
      • Kretzschmar I.
      • Scheuermann J.
      • Neri D.
      • Zoete V.
      • Michielin O.
      • de Pereda J.M.
      • Akhmanova A.
      • Volkmer R.
      • Steinmetz M.O.
      Sequence determinants of a microtubule tip localization signal (MtLS).
      ). Though the core SXIP motifs of +TIPs are indispensable for EB1 binding, the residues surrounding the motif also play important role to further facilitate EB1-binding. Usually, the motif is flanked in between an unstructured region rich with charged, more of basic residues and Serine residues (
      • Honnappa S.
      • Gouveia S.M.
      • Weisbrich A.
      • Damberger F.F.
      • Bhavesh N.S.
      • Jawhari H.
      • Grigoriev I.
      • van Rijssel F.J.
      • Buey R.M.
      • Lawera A.
      • Jelesarov I.
      • Winkler F.K.
      • Wuthrich K.
      • Akhmanova A.
      • Steinmetz M.O.
      An EB1-binding motif acts as a microtubule tip localization signal.
      ,
      • Jiang K.
      • Toedt G.
      • Montenegro Gouveia S.
      • Davey N.E.
      • Hua S.
      • van der Vaart B.
      • Grigoriev I.
      • Larsen J.
      • Pedersen L.B.
      • Bezstarosti K.
      • Lince-Faria M.
      • Demmers J.
      • Steinmetz M.O.
      • Gibson T.J.
      • Akhmanova A.
      A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins.
      ,
      • Buey R.M.
      • Sen I.
      • Kortt O.
      • Mohan R.
      • Gfeller D.
      • Veprintsev D.
      • Kretzschmar I.
      • Scheuermann J.
      • Neri D.
      • Zoete V.
      • Michielin O.
      • de Pereda J.M.
      • Akhmanova A.
      • Volkmer R.
      • Steinmetz M.O.
      Sequence determinants of a microtubule tip localization signal (MtLS).
      ). Ska1 SHLP motif is flanked between several basic amino acids (K82, H86, K88, K106, K117) and Serine residues (S76, S103, S108); and many of them are conserved (Figure 3a). However, whether such unique sequence feature favors Ska1-EB1 binding remains to be tested in the future. Our results implicate that Ska1 recognizes EB1 on the MT plus ends through a molecular process that is analogous to the SXIP-type +TIPs. It is also to be noted that Ska1 is usually found to localize to the plus ends of the KT-targeting MTs, but not other types of MTs (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ,
      • Welburn J.P.
      • Grishchuk E.L.
      • Backer C.B.
      • Wilson-Kubalek E.M.
      • Yates 3rd, J.R.
      • Cheeseman I.M.
      The human kinetochore Ska1 complex facilitates microtubule depolymerization-coupled motility.
      ,
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ,
      • Sivakumar S.
      • Janczyk P.L.
      • Qu Q.
      • Brautigam C.A.
      • Stukenberg P.T.
      • Yu H.
      • Gorbsky G.J.
      The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores.
      ). It is possible that binding with other proteins, such as Ska 2, 3 (
      • Jeyaprakash A.A.
      • Santamaria A.
      • Jayachandran U.
      • Chan Y.W.
      • Benda C.
      • Nigg E.A.
      • Conti E.
      Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface.
      ,
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ,
      • Abad M.A.
      • Zou J.
      • Medina-Pritchard B.
      • Nigg E.A.
      • Rappsilber J.
      • Santamaria A.
      • Jeyaprakash A.A.
      Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain.
      ) and NDC80 (
      • Monda J.K.
      • Whitney I.P.
      • Tarasovetc E.V.
      • Wilson-Kubalek E.
      • Milligan R.A.
      • Grishchuk E.L.
      • Cheeseman I.M.
      Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces.
      ,
      • Huis In 't Veld P.J.
      • Volkov V.A.
      • Stender I.D.
      • Musacchio A.
      • Dogterom M.
      Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling.
      ,
      • Helgeson L.A.
      • Zelter A.
      • Riffle M.
      • MacCoss M.J.
      • Asbury C.L.
      • Davis T.N.
      Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments.
      ) facilitate such KT microtubule-specific localization. KT microtubule-specific localization is also known for another EB1 binding +TIP, (MCAK) (
      • Montenegro Gouveia S.
      • Leslie K.
      • Kapitein L.C.
      • Buey R.M.
      • Grigoriev I.
      • Wagenbach M.
      • Smal I.
      • Meijering E.
      • Hoogenraad C.C.
      • Wordeman L.
      • Steinmetz M.O.
      • Akhmanova A.
      In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends.
      ).
      In conclusion, our results provide a mechanistic basis for formation of stable microtubule-kinetochore attachment during mitosis and revealed the involvement of a more general MAPs such as EB1 in regulating kinetochore functions through site-specific interaction with the outer KT complex protein Ska1. It is important to note that function of Dam1 complex in yeast, the functional analog of metazoan Ska, is also regulated by EB1 (
      • Dudziak A.
      • Engelhard L.
      • Bourque C.
      • Klink B.U.
      • Rombaut P.
      • Kornakov N.
      • Janen K.
      • Herzog F.
      • Gatsogiannis C.
      • Westermann S.
      Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.
      ) through a similar conserved motif of Dam1 protein, Duo1. These suggest a wider implication of our results. As EB1 is involved in organizing numerous plus end targeting cargo proteins at the MT plus ends, a process that is likely to generate diverse plus end structures; It will be interesting to characterize the molecular details of those structures and their functional involvement in chromosome segregation.

      Experimental Procedures

      Reagents and Antibodies

      Thymidine, DAPI, GTP, PIPES, nocodazol and EGTA were obtained from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from, Thermo Fisher Scientific (Massachusetts, U.S.A). Tetracycline-free FBS was obtained from Cytiva (Massachusetts, U.S.A). Mouse monoclonal antibodies against EB1 (Cat # 610534), Actin (Cat# 612656) were obtained from BD Biosciences (California, U.S.A). Mouse monoclonal Myc (Cat# 66004-1-1g), and C-Myc (Cat# Sc-40), antibodies were obtained from Proteintech (Rosemont, U.S.A) and Santa Cruz Biotechnologies, (CA, U.S.A), respectively. Rabbit polyclonal antibody of Ska1 (Cat # ab118586), Ska3 (Cat # ab186003) and rat monoclonal antibody of EB1 (Cat # ab53358) were obtained from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibody for Ska1 (NBP1-72131) was obtained from Novus Biologicals (Centennial U.S.A). Mouse monoclonal antibody against α-tubulin (Cat # T6199) and the rabbit polyclonal anti-EB1 (Cat # E3406) and anti-GST (Cat # G7781) were obtained from Sigma (St. Louis, MO). Mouse monoclonal antibodies of Hec1 (Cat # sc-135934) and CENP-A (GTX13939) were obtained from Santa Cruz Biotechnologies, (CA, U.S.A), and Genetex (CA, U.S.A), respectively. GFP antibody (Cat # 632381) was obtained from Clontech, Takara (U. S. A.). GFP trap beads (gt-20) were obtained from Chromotech (Germany). The dilutions of the primary antibodies were: EB1 (IF-1:1000 of Sigma, Abcam and WB-1:3000 of BD, Sigma), α-tubulin (IF- 1:700 and WB:-1:3000), Ska1 (IF-1:250 Abcam, WB- 1:500 Novus), Hec1 (IF-1:200), CENP-A (IF -1:200), Myc (IF-1:300, WB-1: 500. Alexa fluor conjugated donkey anti-mouse 488 and anti-rabbit 568 secondary antibodies were obtained from Invitrogen (CA, U.S.A). Anti-rat TRITC, Anti-mouse Cy5, and peroxidase-conjugated secondary antibodies were obtained from Jacksons Immuno Research (PA, U.S.A.).

      Cell culture and transfection

      HeLa and HEK 293T cells were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1.5 mg/ml sodium bicarbonate, 100 μg/ml penicillin and 100 μg/ml streptomycin in a humidified environment with 5% Co2. For depletion of endogenous Ska1, 60% confluent HeLa cells were transfected with Ska1 siRNA (5′-CCCGCTTAACCTATAATCAAA-3′) (Cat # D-015917-04)(
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ). For control, Sigenome siRNA (5′-GCCAUUCUAUCCUCUAGAGGAUG-3′) (Cat # D-001210-01-05) was used. For rescue experiments, siRNA resistant GFP-tagged Ska1 WT and various mutant variant plasmid DNA was transfected after 12 hrs of siRNA treatment. Cells were treated with MG132 (25 μM) for 2 hrs prior to collection 48 hrs post transfection of the plasmid DNA. Inducible Cas9 HeLa cells stable for specific EB1 guide RNA were obtained from Iain Cheeseman (Whitehead Institute, MIT, USA). The cells were maintained in growth medium containing 10% tetracyclin-free FBS (Cytiva, USA). For generating EB1 knockout, Cas 9 was induced by treating the cells with 2 μg/ml doxycyclin at 24 hrs interval for four days prior to analysis of EB1 protein level (
      • McKinley K.L.
      • Cheeseman I.M.
      Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects.
      ). For rescue experiments with Myc-Ska1 1-132 GFP, the cells after two days of doxycyclin treatment were transfected with Ska1 siRNA (12 hrs) followed by transfection of the plasmid DNA while continuing the doxycycline treatment for the next two days prior to analysis. For assessing KT localization of Myc-Ska1 1-132 in presence of nocodazole, HeLa cells transfected with Myc Ska1 1-132 under endogenous Ska1 depletion were synchronised by double thymidine and treated with 300 nM nocodazole (6 hrs after thymidine release) for 4 hrs prior to fixing and staining. Lipofectamine RNAimax (Invitrogen Life Technologies) was used as vehicle for transfection of siRNA and lipofectamine 3000 was used for plasmid DNA transfection.

      Plasmids and proteins

      Ska1-GFP construct was made by PCR amplification of wild type Ska1 from pIC291plasmid (Addgene, U. S. A.) and cloned in to pcDNA3-EGFP vector having CMV promoter. The construct was made SiRNA resistant by using site directed mutagenesis. Ska1 1-132 GFP as well as Myc tagged constructs were made by PCR amplification of 1-132 region from Ska1 siRNA resistant construct and was sub cloned in to pcDNA3-EGFP and PCMV-Myc vectors, respectively. WT Ska1-GFP plasmid used in this study was generated using the pIC291 Ska1-GFP (from Iain Cheeseman lab, Whitehead Institute, MIT, USA) as template for PCR amplification of the coding sequence of the human Ska1 gene. The amplified product was ligated into pcDNA3-EGFP (Novagen, Madison, WI, USA) for WT Ska1-GFP. The construct was made Ska1 siRNA-resistant by site directed mutagenesis. Ska1 Δ loop-GFP construct was generated by PCR amplification of the Ska1 region devoid of loop (residues 92-132) and connected from a siRNA resistant PCDNA3.1 Ska1 Δloop-mCherry construct (a gift from A. Jayaprakash, University of Edinburg, U.K.) followed by cloning into a pcDNA3-eGFP vector. The N (1-91)- and C-terminal (133-255) regions were connected through a short peptide (GSSG) in the Ska1 Δloop-mCherry construct (
      • Abad M.A.
      • Medina B.
      • Santamaria A.
      • Zou J.
      • Plasberg-Hill C.
      • Madhumalar A.
      • Jayachandran U.
      • Redli P.M.
      • Rappsilber J.
      • Nigg E.A.
      • Jeyaprakash A.A.
      Structural basis for microtubule recognition by the human kinetochore Ska complex.
      ). Ska1 SHNN-GFP and Ska1ΔSHLP-GFP plasmids were generated by site directed mutagenesis from the wild type Ska1 GFP plasmid. Ska1 1-132 GFP and Myc Ska1 1-132 were generated by PCR amplification of 1-132 region of the WT Ska1-GFP siRNA-resistant construct and was subcloned into pcDNA3-EGFP and pCMV-Myc vectors, respectively. His Ska1-GFP construct was made by PCR amplification of the Ska1-GFP region from pcDNA3 Ska1-GFP plasmid and sub-cloned into pET28a vector. For His Ska1 1-132-GFP, the GFP tagged 1-132 Ska1 region from pcDNA3 EGFP vector containing Ska1 1-132-GFP was PCR amplified and sub-cloned into pET28a vector.
      For GST-tagged EB1, EB1 cloned into a pGEX 5x3 vector was used (
      • Thomas G.E.
      • Bandopadhyay K.
      • Sutradhar S.
      • Renjith M.R.
      • Singh P.
      • Gireesh K.K.
      • Simon S.
      • Badarudeen B.
      • Gupta H.
      • Banerjee M.
      • Paul R.
      • Mitra J.
      • Manna T.K.
      EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.
      ). For obtaining 6xHis Ska1 WT protein, pEC-S-CDF-His Ska1 (gift from A. A. Jeyaprakash, Wellcome Trust Centre for Cell Biology, University of Edinburgh, U.K.) plasmid was used. The His -tagged Ska1 SHNN- and Ska1 ΔSHLP constructs generated from the pEC-S-CDF-His Ska1 through site directed mutagenesis. His-tagged Ska1 Δloop construct was generated by PCR amplification of the Ska1 Δloop region from the Ska1 Δloop-mCherry construct and then subcloned into a pET28a vector. All the His- and GST-tagged plasmids were expressed in E. Coli BL21 DE3 cells and the proteins were purified using Ni2+-NTA (QIAGEN, U.S.A.) and Glutathione Sepharose (GE Healthcare, U.S.A.). The purified proteins were stored at -80 oC. Protein concentrations were estimated using Pierce BCA (Bicinchoninic acid) protein assay kit (Thermoscientific, U.S.A.).

      Immunofluorescence microscopy and image analysis

      Cells after fixing in methanol at -20oC were washed with PBS containing 2% bovine serum albumin and 0.5% Triton X-100. The cells were then incubated with primary antibody for 2 hrs followed by incubation with secondary antibody and DAPI for 60 and 1 min, respectively. Coverslips were mounted using ProLong Gold (Invitrogen, U.S.A.), and the images (63X) were captured using a Leica SP5 laser confocal microscope. The intensity per pixel of Ska1-GFP WT or mutant proteins per kinetochore was measured by selecting regions of interest (ROI) of fixed area around the kinetochore after background subtraction using Image J Fiji software. Chromosome misalignment defects in all cases were quantified in a similar way as previously described (
      • Schmidt J.C.
      • Arthanari H.
      • Boeszoermenyi A.
      • Dashkevich N.M.
      • Wilson-Kubalek E.M.
      • Monnier N.
      • Markus M.
      • Oberer M.
      • Milligan R.A.
      • Bathe M.
      • Wagner G.
      • Grishchuk E.L.
      • Cheeseman I.M.
      The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments.
      ). Briefly, the defects were classified broadly into three classes, Class I, II and III. Class I: a significant number of chromosomes could align at the metaphase plate with a few misaligned chromosomes. Class II: majority of the chromosomes are misaligned and poorly congressed. Class III- severely misaligned chromosomes with multipolar spindles. The sum of class I, II and III represents the total percentage of mitotic cells with congression defects.

      Co-IP and GST pull-down assays

      Cells were mitotically synchronized using double thymidine block. EB1 was immunoprecipitated from the mitotic cell lysates using EB1 antibody(
      • Gupta H.
      • Rajeev R.
      • Sasmal R.
      • Radhakrishnan R.M.
      • Anand U.
      • Chandran H.
      • Aparna N.R.
      • Agasti S.
      • Manna T.K.
      SAS-6 Association with gamma-Tubulin Ring Complex Is Required for Centriole Duplication in Human Cells.
      ). For pull-down of Ska1-GFP or Ska1 mutant -GFP proteins using GFP-trap (Chromotech, Germany), the manufacturer’s protocol was followed. Briefly, the cell lysates were incubated with the equilibrated GFP-trap beads for 4 hrs and then the proteins bound to the beads were analysed after washing the beads followed by lysing with sample buffer. Co-immunoprecipitation of Ska1-GFP proteins was performed using GFP antibody (Takara Bio, Japan).
      Protein A/G beads were used for antibody-based co-immunoprecipitation. In vitro GST pull-down was performed by incubating purified Ska1 WT or mutant proteins with EB1-GST pre-incubated with glutathione-Sepharose beads. The beads were washed with lysis buffer and then boiled in SDS-PAGE sample buffer for immunoblot analysis. For the experiments with peptide aptamer, EB1-GST protein was preincubated with the aptamer for 2 hrs and then the glutathione beads were added. The bead solutions were incubated with the Ska1 WT protein and the pull-down assay was performed.

      Atomic Force Microscopy

      High-speed atomic force microscopy (HS-AFM) images acquired in an in-house built AFM instrument equipped with high-speed recording device (
      • Kodera N.
      • Noshiro D.
      • Dora S.K.
      • Mori T.
      • Habchi J.
      • Blocquel D.
      • Gruet A.
      • Dosnon M.
      • Salladini E.
      • Bignon C.
      • Fujioka Y.
      • Oda T.
      • Noda N.N.
      • Sato M.
      • Lotti M.
      • Mizuguchi M.
      • Longhi S.
      • Ando T.
      Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.
      ,
      • Kodera N.
      • Yamamoto D.
      • Ishikawa R.
      • Ando T.
      Video imaging of walking myosin V by high-speed atomic force microscopy.
      ,
      • Imai H.
      • Uchiumi T.
      • Kodera N.
      Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.
      ). Sample stage consists of a mica sheet of 1.5 mm diameter and ∼ 0.05 mm thickness attached onto a glass cylinder (2 mm height and 2 mm diameter) through epoxy glue. The mica-attached cylinder is sticked onto the Z piezo of the scanner by using nail polish. Freshly cleaved mica surface was prepared by peeling off the top layer of mica sheet using an adhesive tape. 2 μl of protein (EB1 or Ska1) in BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) with varying concentrations (10 to 30 nM) was loaded onto the mica sheet and incubated for 3 min. The stage containing the sample was then rinsed with 20 μl of BRB80 buffer to remove the floating samples and then was immersed in a liquid cell containing the observation buffer. AFM images were then captured in tapping mode using small cantilevers (BL-AC10DS-A2, Olympus) (resonance frequency, ∼ 0.5 MHz in water, quality factor, ∼ 1.5 in water, Spring constant, ∼ 0.1 N/m). The cantilever’s free oscillation amplitude A0 and set-point amplitude As, were set at 1 to 2 nm and 0.9 ∼ 0.9 x A0, respectively. Images were captured at 150 milli-second per frame.
      HS-AFM images were analyzed and refined by using laboratory-made software (
      • Kodera N.
      • Noshiro D.
      • Dora S.K.
      • Mori T.
      • Habchi J.
      • Blocquel D.
      • Gruet A.
      • Dosnon M.
      • Salladini E.
      • Bignon C.
      • Fujioka Y.
      • Oda T.
      • Noda N.N.
      • Sato M.
      • Lotti M.
      • Mizuguchi M.
      • Longhi S.
      • Ando T.
      Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.
      ). Spike noise in the images was removed by applying a low pass filter. XY plane was flattened by using a flattening filter. The XY coordinate of the highest point on the protein domains was determined semi-automatically. First, a point likely to be the highest point was manually assigned and then, the software finds the exact maximum height point in a 5 x 5 pixels area surrounding the manually selected point. The height of individual domains was determined by subtracting the average height of the substrate from the highest identified in the same manner and the distance between the individual domains was measured from the XY coordinates of the highest point.

      Microtubule sedimentation assay

      Tubulin (15 μM) was polymerized in BRB80 buffer (80 mM pipes, 1mM EGTA, 1mM Mgcl2 pH 6.9) in the presence of 10% DMSO, 15 μM Taxol and 1mM GTP at 35 °C for 15 minutes. Aliquots of polymerized MTs were incubated with EB1 (1 μM) for 5 minutes followed by incubation with wild type Ska1 or Ska1ΔSHLP (0.5 μM) for another 15 minutes at room temperature. The MT-protein mixtures were then fixed with 1% glutaraldehyde in BRB80 for 5 minutes at room temperature followed by diluting 50 times in BRB80 buffer prior to layering on a 15% glycerol cushion and sedimentation onto 0.1% poly-L-lysine-coated coverslips. Coverslips were blocked with 1% BSA-BRB80 for 30 minutes and incubated with mouse monoclonal α-tubulin (Sigma), rat monoclonal EB1 (Abcam) and rabbit polyclonal Ska1 antibodies (Novus) for 45 min followed by incubation with secondary antibodies, anti-mouse Alexa 555, anti-rat Alexa 488, and anti-rabbit Alexa 647. Images were captured using Leica SP5 laser confocal microscope. The fluorescence intensities of the proteins associated with MTs were analyzed using Leica LAS AF lite software. The per-pixel intensity of Ska1 bound to MTs was quantified by drawing line ROI of 2 μm length on the MTs (∼120 in number in three experiments) after background subtraction.

      Synthesis and purification of Ska1 peptide

      Fmoc-based solid state peptide chemistry was used for the synthesis of Ska1 SHLP peptide (Ska1 p) (amino acid sequence-KENVPSHLPQVTVT) using PS3TM Peptide Synthesizer. Fmoc protected amino acids and the reagents were purchased from sigma Aldrich. Rink amide MBHA resin (Novabiochem, Germany) was used as the solid surface for the attachment of C-terminal amino acid of the peptide. Deprotection of the N-alpha position take place next followed by the activation and coupling of the second amino acid. HBTU ((2-(1H-benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate) was used as the coupling agent. The steps were repeated till the last amino acid and acetic anhydride was used to acetylate the N-terminus of the peptide. Resin was then washed using dichloromethane (DCM) and dried. Reaction using the cleavage cocktail (88 % trifluoroacetic acid (TFA), 5 % phenol, 5 % water and 2 % tri-isopropyl silane (TIPS) cleaved the peptide from the resin. Cleaved peptide was then precipitated in ice cold ether, dried and dissolved in glacial acetic acid to lyophilize. Reverse phase high-performance liquid chromatography (HPLC) (Agilent Technologies, U.S.A.) was used to purify the peptide. Purity was confirmed using matrix-assisted laser desorption/ionization mass spectrometry. Pure peptide was then concentrated washed and lyophilized in water.

      NMR Titration experiments

      All the 15N-1H TROSY titration experiments were carried out in 700 MHz NMR spectrometer with 16 scans and 256 complex points. To determine the interaction between EB1 and Ska1 peptide, increasing concentrations of Ska1 starting from 0.5 μM to 65 μM was added to 95 μM 2H-15N labelled EB1 in 50 mM potassium phosphate buffer containing 300 mM KCl, 1 mM DTT and 10 % D2O at pH 6.8. All the 15N-1H TROSY spectra were processed using NMRPIPE(
      • Delaglio F.
      • Grzesiek S.
      • Vuister G.W.
      • Zhu G.
      • Pfeifer J.
      • Bax A.
      NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
      ) and SPARKY(
      • Lee W.
      • Tonelli M.
      • Markley J.L.
      NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy.
      ) software.

      Chemical shift perturbation (CSP)

      The combined change in chemical shift on 15N and 1H dimension was calculated using the equation
      Δδ = √ (ΔδHN) 2 + (0.17∗ΔδN) 2
      Δδ = combined chemical shift in Hz
      ΔδHN = chemical shift change in the 1H dimension (Hz)
      ΔδN = chemical shift change in the 15N dimension (Hz)

      Statistical Analysis

      Data are presented as mean +/- SEM. The normally distributed data were analyzed with modified Student’s (Welch) t test at the 99% confidence level. Wherever applicable, one way ANOVA followed by Tukey’s multiple comparison tests were performed. The data were plotted and analyzed using Origin Pro 8.6, and GraphPad Prism 6 software. The figures were organized using Adobe Photoshop and Adobe Illustrator.

      Data availability statement

      All relevant data are available.

      ACKNOWLEDGEMENTS

      We thank Prof. Toshio Ando and Dr. Wei Weilin for technical support of HS-AFM. We thank Iain Cheeseman, Whitehead Institute, MIT, U.S.A. for providing CRISPR-Cas9-based EB1 knock-out cells and Ska1 plasmid. We also thank A.A. Jeyaprakash, Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, U.K. for providing Ska1 deletion constructs. This work was partly supported by Extramural Collaborative Research Grant of Cancer Research Institute, Bio-SPM, Kanazawa University, Japan (TKM). Financial supports from DBT, Govt. of India and DST-SERB, Govt. of India to TKM are thankfully acknowledged.

      Supplementary data

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