Two-Dimensionality of Yeast Colony Expansion Accompanied by Pattern Formation

<div><p>Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the <i>FLO11</i> gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that <i>FLO11</i> gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of <i>FLO11 wild type</i> (<i>FLO11</i>) cells confers a fitness advantage over <i>FLO11 knockout</i> (<i>flo11</i>Δ) cells on the agar surface.</p></div

<i>FLO11</i>-induced wrinkles on the colony surface.

<p><i>(A, D)</i> Frozen blocks oriented across-spokes (blue box indicating estimated location) and radially (yellow box indicating estimated location) were cut from the <i>FLO11 (A)</i> or <i>flo11Δ (D) S. cerevisiae</i> colonies on 1.5% agar and 1.0% glucose YPD plates. The scale bar was 7500 µm. <i>(B)</i> Montage of the cryosections oriented across-spokes of a <i>FLO11</i> colony <i>(A)</i> indicating wrinkles (red box) and spokes (green box). <i>(C)</i> Radial cryosectioning of the <i>FLO11</i> colony <i>(A). (E, F)</i> Montage of the cryosections oriented across-spokes <i>(E)</i> or radially <i>(F)</i> for a <i>flo11Δ</i> colony <i>(D)</i>. The scale bar <i>(B, C, E, F)</i> was 500 µm. <i>(G)</i> A schematic showing primary wrinkle formation (red dashed line), the saturation of which upon increasing stress leads to secondary wrinkle formation (green dashed line). The agar substrate on which the colony expands is shown in blue.</p

Pattern forming <i>FLO11 S. cerevisiae</i> cells out-expand <i>flo11Δ</i> cells during head-to-head competition.

<p><i>(A, B, C)</i> Schematic showing the range expansion of mixed populations segregated into sectors with constant or gradually changing sector angles along the radius due to fitness differences between the particular sector and the adjacent sectors. <i>(A)</i>: Neither population has advantage. <i>(B)</i>: Unlabeled population has advantage. <i>(C)</i>: Red-labeled population has advantage. <i>(D, E)</i> A small competitive advantage of unlabeled cells is observed between isogenic cells of unlabeled and mCherry-labeled <i>flo11Δ</i> cells. <i>(F, G)</i> A small competitive advantage of unlabeled cells is observed between unlabeled and mCherry labeled <i>FLO11</i> cells. <i>(H, I)</i> Unlabeled <i>FLO11</i> cells out-expanded mCherry-labeled <i>flo11Δ</i> cells with a conspicuous increase in the unlabeled sector angle, in comparison to minimal competition between isogenic cells (see below). <i>(J, K)</i> Reverse labeling of <i>(H, I)</i> showed that mCherry-labeled <i>FLO11</i> cells overtook the mixed colonies after some time, despite of the initial lack of expansion advantage against unlabeled <i>flo11Δ</i> cells. Bright field <i>(D, F, H, J)</i> and mCherry <i>(E, G, I, K)</i> were shown respectively. Contrast is adjusted in Adobe Photoshop CS for mCherry images. All cells were grown on 1.0% agar, 0.5% galactose YPGal plates.</p

Colony size and irregularity for various glucose and agar concentrations.

<p><i>(A, B)</i> Images of <i>FLO11 (A)</i> and <i>flo11Δ (B)</i> on YPD plates containing different glucose (0.5%, 1.0%, 2.0%) and agar (1.5%, 3.0%, 6.0%) concentrations around day 20. <i>(C, D)</i> The expansion of colony area <i>(C)</i> and the irregularity <i>(D)</i> of <i>FLO11</i> (red curves) and <i>flo11Δ</i> (green curves) colonies over the 60-day time course. <i>FLO11</i> colonies (red curves) demonstrated higher maximum colony size <i>(C)</i> with higher irregularity <i>(D)</i> at the colony rim than the <i>flo11Δ</i> colonies (green curves) in all conditions tested. The maximum colony size <i>(C)</i> of both <i>FLO11</i> (red curves) and <i>flo11Δ</i> (green curves) colonies increased with glucose and inversely depended on agar concentrations. The irregularity of <i>FLO11 (D)</i> (red curves) inversely depended on both the agar and the glucose concentrations, compared to the minimal irregularity of <i>flo11Δ (D)</i> (green curves) colonies throughout the time course at all conditions tested. Thinner curves represent different replicates while thicker curves represent their average up until all the replicates were present.</p

Mathematical model of colony expansion.

<p><i>(A–C)</i> A snapshot of the colonies at the end of simulation. Although these simulations are started with circular colonies, over time petals appear. The color scale represents cell density (arbitrary units). <i>(D–F)</i> The maximum colony area is higher upon higher initial glucose concentration, in agreement with the experimental results in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003979#pcbi-1003979-g001" target="_blank">Fig. 1</a>. The dimensionless “colony area ratio” was the ratio of colony area to the area of simulation box, and glucose concentration corresponded to the initial value of glucose in the simulation, and was chosen as a constant over space. Time is a rescaled variable measured in arbitrary units. <i>(G–I)</i> Simulated colony irregularity (P2A) plotted as a function of time. Similar to experiments (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003979#pcbi-1003979-g001" target="_blank">Fig. 1</a>), in our model P2A is initially at a basal level and then increases abruptly to a large value. This increase in P2A corresponds to petal formation and occurs as a result of competition over glucose among cells that make up the colony rim. Interestingly, the maximum value of P2A decreases with increasing glucose levels. This result is likely due to decreased intercellular competition over nutrients in the early stages of expansion and is compatible with experiments in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003979#pcbi-1003979-g001" target="_blank">Fig. 1</a>, where colonies exhibit less structure as glucose levels increase.</p

Analysis of the wavelengths of the colony surface patterns.

<p><i>(A)</i> The wavelength of spokes (₂) measured manually and by Fast Fourier Transformation (FFT) decreased with increasing agar density, while the wavelength of primary wrinkles (₁) from manual measurements were less sensitive to the change of agar density. Since the automated FFT measurement was sensitive to noise in the image, FFT did not detect the small-wavelength primary wrinkles as significant. The wavelength of the wrinkles was shorter than that of the spokes at all agar levels tested. The experimentally measured geometric mean wavelength of spokes matched best the ESVS thick substrate model, while the geometric mean wavelength of primary wrinkles matched the ESVS thin substrate model. The inserted image shows the primary and secondary wrinkles measured at the outer radii, indicated by red and green lines, respectively. <i>(B)</i> The Fourier wavelengths plotted as a function of section radius, independent of time for <i>FLO11</i> colony at 0.5% glucose and 0.9% agar. <i>(C)</i> Same as <i>(B)</i>, but for a <i>flo11Δ</i> colony at 0.5% glucose and 0.9% agar. Each dot <i>(C, D)</i> represented a colony from a replicate and a time point. <i>(D)</i> The percent of significant points (Fourier spectral peak height>1.5) plotted as a function of agar. The cell type (<i>FLO11</i> or <i>flo11Δ</i>) is indicated in white font inside the circles.</p