Table S2 from The Ndc80 complex targets Bod1 to human mitotic kinetochores

Kinetochore and centromeric proteins detected as interactors of Bod1-GFP in mitotic HeLa cell

Supplementary figures S1-S5 from The Ndc80 complex targets Bod1 to human mitotic kinetochores

Supplementary figures and associated figure legend

RtkS (SMDB11_2269) is a post-translational activator of T6SS activity.

<p>(A) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>rtkS</i>, Δ<i>ppkA</i>Δ<i>rtkS</i> and Δ<i>pppA</i>Δ<i>rtkS</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (B,D) T6SS-dependent anti-bacterial activity as determined by recovery of target organisms <i>P</i>. <i>fluorescens</i> or <i>S</i>. <i>marcescens</i> ATCC274 following co-culture with strains of <i>S</i>. <i>marcescens</i> Db10 (mutants as above, together with Δ<i>tagF</i>Δ<i>rtkS</i>, Δ<i>ppkA</i>Δ<i>tagF</i> and Δ<i>ppkA</i>Δ<i>tagF</i>Δ<i>rtkS</i>, as indicated). Individual data points are overlaid with mean +/- SEM. (C) Representative images of wild type (WT) and Δ<i>rtkS</i> mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions. In the lower three panels these strains are further carrying the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of RtkS <i>in trans</i> (+ RtkS, pSC590). Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Scale bar, 5 μm.</p

Phosphorylation of Fha promotes formation of Fha and TssB foci in <i>S</i>. <i>marcescens</i> Db10.

<p>(A) T6SS-dependent secretion of Hcp by wild type (WT) <i>S</i>. <i>marcescens</i> Db10 or mutants lacking the kinase PpkA (Δ<i>ppkA</i>) or the phosphatase PppA (Δ<i>pppA</i>), and by derivatives expressing fusions of GFP to the C-terminus of TssB (TssB-GFP) and mCherry to the C-terminus of Fha (Fha-mCherry), in an otherwise wild type, Δ<i>ppkA</i> or Δ<i>pppA</i> mutant background. The T6SS inactive mutant Δ<i>tssE</i> is a negative control and cellular and secreted fractions were subjected to immunoblotting using anti-Hcp antisera. (B) T6SS-dependent anti-bacterial activity of fluorescent reporter strains against a <i>P</i>. <i>fluorescens</i> target. Number of recovered target cells following 4 h co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10 as indicated. Individual data points are overlaid with mean +/- SEM (n = 4). (C) Representative images of cells expressing TssB-GFP and Fha-mCherry fusions, in a wild type, Δ<i>ppkA</i> or Δ<i>pppA</i> mutant background. From left to right, panels show individual fluorescence channels (TssB-GFP, Fha-mCherry), an overlay of the fluorescent channels (Merge; GFP signal false-coloured green and mCherry red), and the corresponding DIC image. Inset panels show a zoomed-in view of the region represented by the white box; for the Δ<i>ppkA</i> mutant, the contrast has been increased in the inset panels to reveal non-focal TssB-GFP fluorescence. Scale bars, 5 μm. (D) Fluorescent signal intensity measurement of the segmented TssB-GFP signal in wild type, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>tagF</i> and Δ<i>ppkA</i>Δ<i>tagF</i> mutants (au, arbitrary fluorescence units). The boundaries of the boxes represent the 25 and 75 percentiles and the horizontal lines are the median values of the analysed population. The whisker bars represent standard error (10 and 90 percentiles). Regions of interest from at least 32 fields of view were analysed in each case. Relative frequency of TssB-GFP foci (expressed per cell at a single timepoint) were estimated by combining automated focus detection with manual cell counting in a representative 15 of these images.</p

Overexpression of TagF inhibits formation of TssL-mCherry foci.

<p>Representative images of wild type (WT) <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and TssL-mCherry fluorescent fusions and carrying either the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of <i>tagF in trans</i> (+ TagF, pSC701). Panels show individual fluorescence channels (TssB-GFP, TssL-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Scale bar, 5 µm.</p

Loss of PppA limits spatial redistribution of the T6SS apparatus.

<p>(A) Localisation of TssB-GFP in the wild type or Δ<i>pppA</i> mutant of <i>S</i>. <i>marcescens</i> Db10 was observed over a 30 min period, with image acquisition every 90 s, and a representative time-lapse sequence of each is shown. The fluorescent signal in individual frames is colour coded by time according to the scale shown in part B and the frames are superimposed. Left and centre image panels, fluorescence projections with the region indicated by the white box in the centre panel being magnified in the left panel; right image panel, corresponding DIC image. The yellow arrow indicates an example of a white coloured focus of TssB-GFP readily visible in composite images from the Δ<i>pppA</i> strain. A further seven fields of view for each strain are presented in Supporting Information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007230#ppat.1007230.s003" target="_blank">S3 Fig</a>. (B) Schematic illustration of how fluorescence projection images were generated. (C) Magnified sections from individual frames of the same time-lapse sequences as part A. First and last panels show GFP signal false-coloured in green and overlaid on the corresponding DIC image of the microcolony, other panels show the TssB-GFP fluorescence signal from the area indicated by the white boxes. The acquisition time in seconds is indicated. (A, C) Scale bar, 5 μm. The full time-lapse sequences including DIC are presented in Supporting Information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007230#ppat.1007230.s004" target="_blank">S4 Fig</a>.</p

TagF is a negative regulator whose overexpression inhibits T6SS activity.

<p>(A) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>tagF</i>, Δ<i>ppkA</i>Δ<i>tagF</i>, Δ<i>pppA</i>Δ<i>tagF</i> and Δ<i>fha</i>Δ<i>tagF</i>) strains of <i>S</i>. <i>marcescens</i> Db10, carrying either the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of <i>tagF in trans</i> (+TagF, pSC701). (B) T6SS-dependent anti-bacterial activity as determined by recovery of target organism <i>P</i>. <i>fluorescens</i> following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10, carrying vector control or <i>tagF</i> expression plasmid. Individual data points are overlaid with mean +/- SEM (n = 4). (C) Representative images of wild type and Δ<i>pppA</i> mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions and carrying either vector control or <i>tagF</i> expression plasmid. Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Scale bar, 5 μm.</p

Schematic model for post-translational regulation of T6SS firing in <i>S</i>. <i>marcescens</i>.

<p>Prior to assembly of the T6SS basal complex (membrane complex, TssJLM, plus baseplate complex, TssEGFK-VgrG), the membrane complex components are free to move around the cell. TagF inhibits assembly of the mature membrane complex which represents the site of baseplate docking. In response to an unknown signal, RtkS interacts with the periplasmic domain of PpkA, leading to PpkA autophosphorylation and phosphorylation of Fha, which is antagonised by PppA. Phosphorylated Fha multimerises and associates with the T6SS, overcoming TagF-mediated repression of basal complex formation. Next, the inner Hcp tube and surrounding TssBC sheath assemble on VgrG and TssE, respectively, and extend into the cytoplasm. This ‘extended’ T6SS is primed and ready to fire. During the firing event, the TssBC sheath contracts, propelling the Hcp-VgrG-PAAR puncturing structure out of the cell through the membrane complex and into a target cell. Effectors decorating the Hcp-VgrG-PAAR structure are released into a target cell, or else to the medium if no target is present. TssH then binds the contracted TssBC sheath and initiates depolymerisation, the basal complex disassembles and PppA dephosphorylates Fha. For a new round of firing to occur, in a wild type cell, TagF-mediated repression must again be overcome by phosphorylation of Fha, allowing time for the T6SS components to move and reassemble in a new spatial location. In the absence of PppA, Fha remains fully phosphorylated, leading to a tendency for the T6SS to immediately reassemble in the same place. When PpkA and TagF are both absent, the intrinsic ability of the system to assemble is observed but anti-bacterial activity is less efficient than with proper post-translational activation in place. IM, inner membrane; PP, periplasm; OM, outer membrane.</p

RtkS interacts with the periplasmic domain of the kinase PpkA, promoting its stability and consequent phosphorylation of Fha.

<p>(A) Bacterial two-hybrid assay to detect interactions between the periplasmic domain of PpkA (PpkA^P; amino acids 363–482) and mature RtkS (RtkS; amino acids 20–328), each fused with T18 or T25 (PpkA^P-T18, pSC593; T25-PpkA^P, pSC594; RtkS-T18, pSC591; T25-RtkS, pSC592). Negative controls were provided by the empty vectors, pUT18 and pT25 (-), and a positive control by the self-interaction of TssK (+ve; TssK-T18, pSC048, and T25-TssK, pSC053). Shown is the β-galactosidase activity, expressed as Δ405/min/ml/OD₆₀₀, of the reporter strain transformed with the combinations of plasmids indicated. Bars show mean +/- SEM (n = 3 independent transformations). (B) Co-purification of RtkS and PpkA under native conditions. Total membrane fractions of wild type <i>S</i>. <i>marcescens</i> Db10 (no tag) or strains expressing PpkA with a C-terminal HA tag (PpkA-HA), RtkS with a C-terminal His₆ tag (RtkS-His), or both, from the normal chromosomal location were subjected to anti-HA immunoprecipitation (top panels) or His affinity purification using Ni²⁺-NTA (bottom panels). Bound proteins in each case were separated by SDS-PAGE and subjected to anti-HA immunoblot (to detect PpkA-HA) or anti-His₆ immunoblot (to detect RtkS-His), as indicated. (C) Levels of PpkA in the presence or absence of RtkS were determined by immunoblot detection of PpkA-HA in an otherwise wild type background (PpkA-HA) or in the Δ<i>rtkS</i> mutant (PpkA-HA, Δ<i>rtkS</i>), and in strains carrying the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of RtkS <i>in trans</i> (+ RtkS, pSC590). PpkA-HA levels were measured in total protein samples, with EFTu (bottom panel) representing a loading control and 0.5x the amount of total protein loaded in the right hand most lane compared with the other samples, as indicated. (D) Quantification of Fha phosphorylation by detemination of the levels of Fha phosphopeptide in otherwise wild type (WT) and mutant (Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>rtkS</i>) strains of <i>S</i>. <i>marcescens</i> Db10 expressing Fha-HA from the normal chromosomal location. Levels of phosphopeptide were determined by label-free mass spectrometry and individual data points are shown, with the mean indicated by a line. Levels of phosphopeptide were quantified in 3/3 replicates for WT and Δ<i>pppA</i>, and in 2/3 replicates for Δ<i>rtkS</i>. The phosphopeptide was not detected in the Δ<i>ppkA</i> mutant.</p

Phosphorylation of Fha and formation of Fha foci is no longer required for T6SS activity in the absence of the post-translational regulator TagF.

<p>(A) Schematic depiction of the T6SS gene cluster of <i>S</i>. <i>marcescens</i> Db10 showing the genes encoding components of the T6SS post-translational regulatory system in this organism (in red). Core T6SS components are shown in blue, with letters indicating TssA-M, and effector/immunity pairs are shown in purple/pink. (B) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>fha</i>, Δ<i>tagF</i>, Δ<i>ppkA</i>Δ<i>tagF</i>, Δ<i>pppA</i>Δ<i>tagF</i> and Δ<i>fha</i>Δ<i>tagF</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (C) T6SS-dependent anti-bacterial activity as determined by recovery of target organism <i>P</i>. <i>fluorescens</i> following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10. Individual data points are overlaid with mean +/- SEM (n = 4). (D) Representative images of wild type and mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions. Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Additionally, TssB-GFP was monitored in the Δ<i>fha</i>Δ<i>tagF</i> background. The mCherry channel image in this case illustrates the low level but frequently observed background signal unrelated to mCherry expression. Scale bar, 5 μm.</p