Motility enhancement through surface modification is sufficient for cyanobacterial community organization during phototaxis.

The emergent behaviors of communities of genotypically identical cells cannot be easily predicted from the behaviors of individual cells. In many cases, it is thought that direct cell-cell communication plays a critical role in the transition from individual to community behaviors. In the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803, individual cells exhibit light-directed motility ("phototaxis") over surfaces, resulting in the emergence of dynamic spatial organization of multicellular communities. To probe this striking community behavior, we carried out time-lapse video microscopy coupled with quantitative analysis of single-cell dynamics under varying light conditions. These analyses suggest that cells secrete an extracellular substance that modifies the physical properties of the substrate, leading to enhanced motility and the ability for groups of cells to passively guide one another. We developed a biophysical model that demonstrates that this form of indirect, surface-based communication is sufficient to create distinct motile groups whose shape, velocity, and dynamics qualitatively match our experimental observations, even in the absence of direct cellular interactions or changes in single-cell behavior. Our computational analysis of the predicted community behavior, across a matrix of cellular concentrations and light biases, demonstrates that spatial patterning follows robust scaling laws and provides a useful resource for the generation of testable hypotheses regarding phototactic behavior. In addition, we predict that degradation of the surface modification may account for the secondary patterns occasionally observed after the initial formation of a community structure. Taken together, our modeling and experiments provide a framework to show that the emergent spatial organization of phototactic communities requires modification of the substrate, and this form of surface-based communication could provide insight into the behavior of a wide array of biological communities

Computational determination of the phase space of collective motility behaviors.

<p>A) Simulations grouped into classes as a function of dimensionless bias force and mean cellular concentration, resulting in the phase diagram shown on logarithmic axes. Colored circles represent parameters with a sufficient degree of directed movement and cell front instability to result in simulations with finger-like morphologies. Different shades correspond to the values of the bias force. In the simulations represented by the grey circles, cells move in a directed fashion toward the light source but do not spontaneously gather into groups due to high cell density, and thus high local EPS production. For the simulations represented by the black circles, little to no cellular movement resulted due to insufficient EPS production and/or bias force. Numbers correspond to the frames in (B). B) Representative morphologies at the end of simulations with parameters from different regions of the phase diagram. In numbered frames, cells are shown in green, EPS is shown in red, and orange indicates co-localization of cells and EPS. The simulations in frames 1–4 have finger-like morphologies with varying group sizes and spacing. The simulation depicted in frame 5 has a moving but uniform cell front (i.e., no distinct groups), while frame 6 reflects a simulation of essentially non-motile cells.</p

Scaling laws reveal simple relationships among cell concentration, light bias, and community morphology.

<p>A) The number of cells in each motile group as a function of initial cellular concentration follows an approximately linear relationship independent of bias force (gray dashed line). B) The mean speed of cellular groups approximately follows a 1/3-power law. C) The ramp time associated with the formation of finger-like morphologies follows an approximately inverse square-root power law. D) The number of cells in each finger and the speed toward the light source are positively correlated for all bias forces. In all panels, colors refer to the bias force legend and correspond to dots in the phase diagram of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003205#pcbi-1003205-g006" target="_blank">Fig. 6A</a>.</p

Cells secrete an extracellular substance that enhances their motility.

<p>A) Typical fingering of a <i>Synechocystis</i> community toward a light source during a phototaxis assay. The contrasted inset shows EPS deposited by motile groups. B) Phase-contrast microscopy of motile groups immediately before, and then at specified intervals after a change in the light direction (orange arrows). The white dashed lines indicate the bounds of a region through which a finger recently passed. The motile group changes course after a change in the light direction, and upon intersection with the EPS trail of a neighboring finger, group speed toward the light increases and the cells become more dispersed. C) Median velocity of cells in the motile group in both the <i>x</i> and <i>y</i> directions with respect to the coordinate system shown in (B); dashed horizontal lines indicate 95% confidence intervals. Left: prior to the change in light direction, cells have positive velocity toward the light source (top), and approximately zero net velocity perpendicular to the light (bottom). Middle: following the change in light direction, the cells reorient and velocity in the <i>x</i> direction rises to a value comparable with the <i>y</i> velocity prior to the light change (bottom), while the net y velocity approaches zero (top). Right: when the group of cells merges with the trail of the neighboring finger (dashed vertical lines), the spread in <i>y</i> velocities increases (top) and the median <i>x</i> velocity increases by approximately three-fold (bottom). D) Histograms of speeds for the same cells (<i>n</i> = 95) before and soon after merging with the trail of secreted extracellular substance, with mean speed and standard deviation indicated in the legend. E) Individual cells experience an increase in speed after the group merges with the trail of another finger, indicating that the change in group dynamics upon merging is coupled to a change in the motility of individual cells.</p

Biophysical model of EPS-based mobility enhancement captures the observed fingering behavior during phototaxis.

<p>A) As shown schematically, each cyanobacterium (green) is assumed to undergo a biased random walk toward the light source (LED). Each cell secretes EPS (blue), whose local concentration increases the cell's mobility <i>M</i> (red). B) The cell deposition, crescent formation, and finger formation observed in experiments (top) are recapitulated by simulations using our model (bottom) with and ; cells are shown in green, EPS in red. Immediately after deposition (left), cells are distributed randomly and the boundary with the substrate is smooth (inset). Cells collect at the edge of the initial deposition area as they migrate toward the light source (middle, 20 hr), with small variations at the interface (inset). The front matures into discrete groups of cells that separately migrate toward the light source (right, 45 hr). Scale bars are 1 mm.</p

EPS decay results in multiple cell fronts and preferred paths for cellular groups.

<p>A) Experimental example of cells splitting into multiple fronts that head toward the light source. B) Phase diagram of the EPS decay model at a constant bias force. In the red region, cells form discrete, motile groups that head toward the light source in one or more cellular fronts. In the gray region, cells move toward the light source but do not form discrete groups. In the black region, cells are essentially non-motile due to insufficient EPS production and/or rapid decay of EPS. C) Late time-point snapshots of simulations from regions of the phase diagram in (B), with a light source at the top (green, cells; red, EPS; orange, colocalization of cells and EPS). Frames 1–4 depict results from simulations in the red region in (B), in which multiple fronts with finger-like morphologies can form similar to (A) with varying group sizes and numbers of distinct cell fronts. Groups of cells behind the primary front often catch up by following EPS trails left by forward groups (inset of frame 2). Frame 5 is from a simulation in the gray region of (B), in which cells move toward the light but do not form discrete groups. Frame 6 depicts the result from a simulation in the black region of (B); the cells are essentially non-motile. Scale bars are 1 mm.</p

Quantitative analysis of community morphologies from a representative simulation.

<p>Using computational image processing at each time point, we quantified the number of fingers and their ramp time, size, and speed. The inset shows the cells (orange), cell front morphologies at evenly-spaced time points (blue contours), the group center positions over time (green lines), and the EPS field at the final time point (grayscale). These data provide a set of quantitative metrics for mapping model parameters to community behaviors.</p

Model successfully predicts effects of increased cellular concentration of finger ramp time and biomass.

<p>Phototaxis time-lapse of <i>Synechocystis</i> cells deposited on an agarose surface with increasing initial concentrations with OD₇₃₀ (from left to right) of 0.6, 0.8, 1.0, 1.2, and 1.4. Time-lapse images were taken at 0, 11, 24, 36, and 49 hr. The images show a negative correlation between initial cell density and time to finger formation, and a positive correlation between initial cell density and number of cells in each finger, both of which are trends predicted by our biophysical model. Scale bar is 1 mm.</p

Biophysical model reproduces key features of group motility during finger merging.

<p>In these simulations, the direction of light bias (white arrows) is rotated 90 degrees between frames 1 and 2. The cells (green) leave an EPS trail (red), and when a finger intersects the EPS trail left by a neighboring finger, the group of cells speeds up and spreads out (blue arrows, frame 3). We observed the same qualitative changes in finger merging experiments (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003205#pcbi-1003205-g002" target="_blank">Fig. 2</a>).</p

<i>Synechocystis</i> cells on a surface accumulate in finger-like projections when moving toward a directional light source.

<p>A) Time-lapse imaging of a <i>Synechocystis</i> community in the presence of a directional light source; images were taken at 0, 11, 24, 36, and 49 hr. White rectangles indicate regions highlighted in (B–F). Scale bar is 1 mm. B) Cells are initially randomly distributed across the surface. (phase contrast image taken at 10× magnification, scale bar is 10 µm.) C) Regions of heterogeneity are evident where the local cell concentrations are slightly higher than in the rest of the initial deposition area. D) Regions of higher cell concentrations start to form small finger-like projections within the initial deposition. E) Cells collect at the edge of the initial deposition and create a ruffled interface, from which finger-like projections will emerge. F) Finger-like projections form as distinct, motile groups of cells that move toward the light source, leaving behind a trail of lower density cells.</p