<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

List of yeast strains.

<p>List of yeast strains.</p

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

<div><p>Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.</p> </div

Time-lapse microscopy of Cdc42-GTP polarization in wild-type a/α diploids.

<p><b>A.</b> A schematics diagram of the bipolar budding pattern. M and D stand for mother and daughter cells, respectively. Red arrows depict the axis of cell polarity. <b>B.</b> Localization of Gic2-PBD-RFP and Cdc3-GFP in diploid wild-type cells (HPY2353). An arrowhead marks Gic2-PBD-RFP localized to the proximal pole in the daughter cell. Numbers indicate time (in min) from the first image. Size bars, 3 µm.</p

Positions of the first bud in a/α daughter cells of wild type and mutants deleted for Cdc42 GAPs.

<p><b>A.</b> Time-lapse DIC images of diploid cells of wild type (YEF473) and <i>rga1Δ</i> (YEF1233). Arrows indicate budding events from daughter cells. Numbers indicate times (in min) from the first image. Size bars, 5 µm. (Histogram) The position of the first bud of daughter cells was scored in wild type (HPY1680), <i>rga1Δ</i> (HPY2205), <i>rga2Δ</i> (HPY2246), <i>bem2Δ</i> (HPY2384), and <i>bem3Δ</i> (HPY2426). The mean percentage ± SD of each budding pattern is shown from three or four independent countings of wild type (n = 106), <i>rga1Δ</i> (n = 144), <i>rga2Δ</i> (n = 56), <i>bem2Δ</i> (n = 108), and <i>bem3Δ</i> (n = 53). Statistical significance was determined by Student's t-test between proximal-pole buddings in wild type and <i>rga1Δ</i> or <i>bem2Δ</i> (marked with asterisks): *p<10⁻⁵ (<i>rga1Δ</i>) and **p = 0.02 (<i>bem2Δ</i>). <b>B.</b> The position of the first bud relative to the birth scar in diploid daughter cells. Cells were double stained with Calcoflour white and WGA-FITC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone.0056665-Frydlov1" target="_blank">[44]</a> from wild type (YEF473), <i>rga1Δ</i> (YEF1233), <i>bud8Δ</i> (YHH415), and <i>rga1Δ bud8Δ</i> (HPY2385). Arrows indicate birth scars. Size bar, 3 µm.</p

Localization of Gic2-PBD-RFP and Cdc3-GFP in <i>rga1Δ</i> cells.

<p><b>A.</b> In <i>rga1Δ</i> cells (HPY2204), Gic2-PBD-RFP localized continuously to (a) the proximal pole or (b) the distal pole from cytokinesis to the next G1 phase. Arrows in (a) & (b) denote the Cdc3 ring splitting and an arrowhead in (a) denotes Gic2-PBD-RFP enriched at the division site (as well as the bud tip). Numbers indicate times (in min) from the first image. Size bars, 3 µm. <b>B.</b> Localization pattern of Gic2-PBD-RFP (red) prior to, during, and after cytokinesis (Cdc3-GFP in green) is summarized from time-lapse imagings of wild type (n = 15), <i>rga1Δ</i> (n = 19), <i>bud8Δ</i> (n = 7) and <i>rga1Δ bud8Δ</i> (n = 8). The proximal-pole localization pattern (marked with 2*) of <i>rga1Δ</i> or <i>rga1Δ bud8Δ</i> daughter cells is different from those seen in wild type and <i>bud8Δ</i> cells (see text for details).</p

Mathematical modeling of Cdc42 polarization.

<p><b>A.</b> A schematic diagram of the reaction-diffusion model with the following parameters: <i>D_m</i> (the diffusion rate coefficient of Cdc42-GDP and Cdc42-GTP on the plasma membrane), <i>k_d</i> (the inactivation rate coefficient of Cdc42 from the GTP- to the GDP-bound states), and <i>k_off</i> (the rate at which the membrane-bound Cdc42-GDP is extracted into the cytoplasm). The function <i>F</i>([<i>cue</i>], [<i>C42T</i>]) represents the Bem1-mediated activation rate; and the function <i>k_R</i>([<i>cue</i>]) is the landmark-signal-dependent recruitment rate of Cdc42 from the cytoplasm to the membrane. See details in Materials and Methods and the values of parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone-0056665-t001" target="_blank"><b>Tables 1</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone-0056665-t002" target="_blank"><b>2</b></a>. <b>Ba.</b> Coordinate of the periphery of an <b>a</b>/α daughter cell. The cell periphery is parameterized by the radial angle (0°–360°) in a clockwise direction starting from the distal pole. <b>Bb</b>. Spatial distributions of the landmark cues and the GTP hydrolysis rate of Cdc42. Both quantities are normalized by their maximal values for better visualization, with the color map scale shown on the right. <b>C.</b> Spatiotemporal dynamics of Cdc42-GTP in a diploid daughter cell. The cell periphery is presented as a 1D vertical axis, with the proximal pole in the middle (180°) and distal pole at the top/bottom (0°/360°). The horizontal axis represents the time window from 0 to 10 min. The localization of Cdc42-GTP at steady state is shown on the 2D cell periphery (right). The level of Cdc42-GTP is normalized by its maximal value at steady state, and the color map is displayed. See parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone-0056665-t001" target="_blank"><b>Tables 1</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone-0056665-t002" target="_blank"><b>2</b></a> for each simulation.</p

Ranges of parameters in the simulations.

<p>Ranges of parameters in the simulations.</p

Localization of Gic2-PBD-RFP and Cdc3-GFP in the diploids homozygous for <i>bud8Δ rga1Δ</i> and <i>bud8Δ</i>.

<p>Imaging was performed as in Fig. 4 except in <i>bud8Δ</i> (HPY2370) and <i>rga1Δ bud8Δ</i> (HPY2371) cells. Arrows denote the Cdc3 ring splitting and arrowheads denote Gic2-PBD-RFP enriched at the proximal pole. Note: Gic2-PBD-RFP became enriched at a site adjacent to the Cdc3 ring in the <i>bud8Δ</i> daughter cell, whereas it appeared within the Cdc3 ring in <i>rga1Δ bud8Δ</i> daughter cell. Numbers indicate times (in min) from the first image. Size bars, 3 µm.</p

Mathematical modeling of Cdc42 polarization in diploid daughter cells deleted for <i>RGA1</i>, <i>BUD8</i> or <i>BUD9</i>.

<p><b>Aa.</b> Coordinates are the same as in Fig. 2 (wild type) but the GTP hydrolysis rate of Cdc42 in the <i>rga1Δ</i> mutant is assumed to be about the same along the perimeter. See parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone-0056665-t002" target="_blank">Table 2</a>. <b>Ab–Ac.</b> Spatiotemporal dynamics of Cdc42-GTP leading to budding at (b) the proximal pole or (c) the distal pole in <i>rga1Δ</i> daughter cells. The horizontal axis represents the time window from 0 to 10 min. The 2D steady-state distribution of Cdc42-GTP is displayed on the right to each simulation. <b>B.</b> Spatiotemporal dynamics of Cdc42-GTP in diploid <i>bud8Δ</i> (top) and <i>bud9Δ</i> (bottom) mutants. The horizontal axis represents the time window from 0 to 20 min. The 2D steady-state distribution of Cdc42-GTP is displayed on the right to each simulation. Note: Cdc42-GTP became polarized at a site adjacent to the center of the proximal pole in <i>bud8Δ</i> (see 20 min time point), unlike in <i>rga1Δ</i> (see Fig. 7A, b).</p