Timing of TEM steps for UM cells.

<p>A) Diagram depicting the steps of TEM used by UM cells. B) Plot of time spent in each phase of intercalation. The data include only cells that eventually disengaged from intercalation; many cells were still intercalated at 12 hrs and could not be assessed for duration of intercalation. Red bars indicate medians.</p

List of yeast strains.

<p>List of yeast strains.</p

<i>BUD2</i> is required for the spindle position checkpoint.

<p><i>arp1Δ GFP-TUB1</i> cells with the additional indicated mutations were assayed for checkpoint integrity by video analysis. Cells with long (late-anaphase) spindles in the mother of a budded cell were followed over time. Checkpoint integrity is the percent of cells in which the spindle that remained intact, i.e. did not break down, for a time greater than the mean plus two standard deviations of the time for normal mitotic exit. A. <i>bud2Δ</i> mutants have a defect in the spindle position checkpoint, with failure to maintain arrest in about half of cells. <i>bub2Δ</i> is a positive control known to have a complete defect. The <i>bud2Δ</i> phenotype does not depend on <i>LTE1</i>, based on the <i>bud2Δ lte1Δ</i> double mutant. <i>BUD6</i> is in a pathway upstream of <i>LTE1</i>, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036127#pone.0036127-Nelson1" target="_blank">[2]</a> and confirmed here. The <i>bud2 bud6</i> double mutant has an exacerbated phenotype, confirming that <i>BUD2</i> is in a genetic pathway independent of <i>BUD6</i> and <i>LTE1</i>. The <i>bud2Δ bud6Δ</i> double mutant does not have a complete loss of phenotype, as <i>bub2</i> does, suggesting a possible third input into the checkpoint control of mitotic exit. B. The bud-site-selection pathway has no role in the spindle position checkpoint. Mutants lacking either Rsr1/Bud1, the only known substrate of Bud2, or Bud5, the GEF for Rsr1/Bud1, have no checkpoint defect. Deleting <i>RSR1/BUD1</i> does not suppress the checkpoint defect of a <i>bud2Δ</i> mutant. <i>bud3</i> and <i>bud5</i> null mutants, defective in axial and all budding patterns, respectively, are also normal.</p

HS1 and TEM of NK cells in transwell assays.

<p>A) Diagram of transendothelial migration assay in a transwell device. B) Depletion of HS1 protein by siRNA, shown by immunoblot after 72 hrs. NK cells were treated with a pool of four siRNAs or one of the four. GAPDH is a loading control. C) Effects of HS1 knockdown on TEM. Plotted values are number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day. Box-and-whisker plots (box: 25th to 75th percentiles, whiskers: min to max, middle line: median). Asterisks indicate statistical significance (*P<0.05. Unpaired Student’s t-test, n = 5 for each condition.) D) Fluorescence micrographs of NK cells, showing expression and co-localization of expressed HS1-tdTomato (red), F-actin (green, Alexa Fluor 488 phalloidin), and total HS1, including endogenous (blue, anti-HS1 staining). E) Fluorescence micrographs of NK cells stained with anti-HS1 to show siRNA-induced depletion of HS1 and expression of siRNA-resistant HS1 protein. F) Expression of siRNA-resistant HS1 in NK cells knocked down for HS1 with siRNA, shown by immunoblot with anti-HS1. Knockdown used a combination of HS1 siRNAs 2 and 3. G) Rescue of TEM phenotype in HS1-knockdown NK cells by expression of HS1. Cells as in panels E and F. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box-and-whisker plots as in panel C. Asterisks indicate statistical significance. (* P< 0.05, *** P < 0.0005. Unpaired Student’s t-test, N = 9–12 experiments for each condition.)</p

Role of Vav1 in TEM by NK cells.

<p>A) Immunoblots with anti-HS1 and anti-Vav1 showing depletion of HS1 and Vav1 after 72 hrs of siRNA treatment. B) Decrease in TEM in transwell assay by NK cells treated with Vav1 siRNA, compared to control siRNA. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. N = 6. Asterisks indicate **P<0.005 (unpaired Student’s t-test). C) Left panel: Immunoblot with anti-HS1. The left lane shows the absence of HS1 in an anti-HS1 immunoprecipitate from a whole-cell lysate of NK cells treated with siRNA targeting HS1. The right lane shows the result for cells treated with control siRNA. Middle panel: Immunoblot with anti-Vav1. The left lane shows the presence of Vav1 protein in an anti-HS1 precipitate from a lysate of NK cells treated with control siRNA. The right lane shows the presence of Vav1 in the lysate. Right panel: Similar to the middle panel, except with a lysate from NK cells depleted for HS1.</p

Expression Rescue of HS1 Mutants in HS1-depleted NK Cells.

<p>A) Phosphorylation of HS1 Tyr397 in response to SDF-1α. Immunoblots probed with anti-Phospho-Tyr397 and anti-HS1. NK cells (5 x 10⁶) treated with SDF-1α (30 ng/mL) for the indicated times (min). B—D) Function of HS1 mutants in TEM by transwell assay. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. B) Mutations of phosphorylated tyrosine residues. Compared to control siRNA (blue), HS1 depletion by siRNA causes decreased TEM (red), and the defect is rescued by expression of wild-type HS1 (green) or siRNA-resistant wild-type HS1 (purple). Expression of siRNA-resistant forms of single-mutant HS1 Y378F (black), single-mutant HS1 Y397F (brown) or double-mutant HS1 Y378F Y397F (dark blue) does not rescue the defect, comparing their values to the value for siRNA-resistant wild-type (purple). Expression of siRNA-resistant HS1 Y222F (orange) rescues with a value that is slightly less, but not statistically significant, from that of siRNA-resistant wild type. Asterisks indicate *P>0.05, **P>0.005 (unpaired Student’s t-test, N = 6–9). C) Mutation of Arp2/3 complex binding site. Expression of siRNA-resistant HS1 with mutation of DDW residues to AAA (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. N = 6 in each case. D) Mutation of SH3 domain at ligand-binding site. Expression of siRNA-resistant HS1 with the mutation W466K (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. n = 6 in each case.</p

Migration Speeds for NK Cells.

<p>Speed (μm/min) defined as net displacement (distance start to finish) divided by time for control vs HS1-depleted cells. The distributions are not Gaussian, so the values listed are median, 95% confidence interval of the median and number of data points (N). P values from two non-parametric tests of significance are listed. Data include tracks for all cells in separate experiments on three different days.</p><p>Migration Speeds for NK Cells.</p

TEM events by NK cells on HDMVEC monolayers based on live-cell movie analysis.

<p>A) Endogenous HS1 (red) and F-actin (green) in NK cells migrating on the surface of HDMVEC monolayer observed by anti-HS1 and phalloidin fluorescence. Scale bar = 20 μm. B) DIC images from a movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.s006" target="_blank">S2 Movie</a>), illustrating how the passage of the NK cell through the endothelial monolayer leaves a defect. Scale bar = 10 μm. C) Speed of cell migration, based on path length. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-1. D</a>) Speed of cell migration, based on net displacement. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-2. E</a>) Percentage of TEM events from movie analysis with SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The difference is not statistically significant by z-test (p = 0.15) or by Fisher’s exact test for a 2 x 2 contingency table (p = 0.19). Data combined from two or three experiments per day on three days. F) Percentage of TEM events from movie analysis without SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The differences between control and depleted-cell values are statistically significant with p values of 0.022, 0.024 and 0.001 for HS-depleted, Vav1-depleted and HS1+Vav1-depleted NK cells, based on chi-square tests with Yates’ correction. No other differences are statistically significant. Data combined from experiments on three days.</p

Effect of Vav1 and HS1 plus Vav1 Depletion on Migration Speeds for NK Cells.

<p>Preparations not treated with SDF-1α. Combination of all tracks from experiments on three days. The distributions are not Gaussian, so the values listed are median, 95% confidence interval of the median and number of data points (N). P values from two non-parametric tests of significance are listed. Data include tracks for all cells in separate experiments on three different days.</p><p>Effect of Vav1 and HS1 plus Vav1 Depletion on Migration Speeds for NK Cells.</p

Transcellular vs paracellular route of TEM.

<p>A) Diagram illustrating paracellular and transcellular routes. B) Representative confocal fluorescence images of cells taking the paracellular (PC) and transcellular (TC) routes. The migrating NK cells appear as small dark holes surrounded by intense anti-ICAM-1 staining, and the endothelial cell-cell junctions are visualized by anti-VE-cadherin staining. The endothelial cell substrate was glass in the upper panel and soft substrate (polyacrylamide) in the lower panel. Scale bar = 10 μm. C) Total number of transendothelial migration events, with the endothelial monolayer on glass or soft substrate (SS). D) Numbers of paracellular and transcellular transmigration events on glass. E) Numbers of paracellular and transcellular transmigration events on soft substrate. For panels D to F, each plotted data point represents the average of three values from one experiment. The mean and standard error of the values of the plotted points are also indicated, by the dotted lines and error bars. HDMVEC cells were washed with SDF-1α-containing media before the addition of NK cells, and NK cells were incubated for 2 hrs on the monolayer before fixation. F and G) Transendothelial migration events and routes. NK cells were incubated for 25 min on the surface of an HDMVEC monolayer. NK cells migrated via transcellular and paracellular routes were counted over entire slide. The data are based on experiments in triplicate on two different days. F) Numbers of events. G) Ratios. The difference in the ratio of transcellular to paracellular events between control and HS1 knockdown is small but statistically significant because the number of data points is large. Based on a 2 x 2 contingency table, Fisher’s exact two-tailed p-value is 0.0024.</p