A Predictive Model for Yeast Cell Polarization in Pheromone Gradients
Fig 2
The response of bar1Δ cells response to homogenous stimulation by α-factor.
A) Schematic of a device to produce a range of pheromone concentrations by using chaotic mixers in dilution chambers. The diagram shows a plan view (left) and cross sections at two magnifications (right). Structured micro channels allow fast mixing and thus permit serial dilutions in a small device. A lectin (concanavalin A) binds yeast cells to the coverslip that forms the roof of the chamber. They receive a constant flow of pheromone, allowing them to be exposed to concentrations that are stable over several hours and can be measured by quantifying the emission of a fluorescent dextran mixed with the pheromone (see Experimental Procedures in S1 Text and Fig A in S1 Text for more details). B) The behavior of cells in micro-channels at various pheromone concentrations. We assessed cell behavior at each pheromone concentration by following cells over time and overlaying differential interference contrast and Spa2-YFP images (see Fig B in S1 Text for images). The graph quantifies the bud/shmoo transition in spatially uniform fields of pheromone and summarizes data from about 4000 cells (on each curve (red and green), at least 160 cells (MP 384) were used to obtain the averages shown for each pheromone dose) in seven independent dilution chambers. The inset shows the standard deviation of the fraction of different events between experiments. The measured dissociation constant of α-factor from Ste2 is indicated (Kd) [41]. The half maximal point of the sigmoidal fit is 1.02 ± 0.03 nM and the Hill coefficient for the transition between budding and shmooing is 6.5 ± 0.6 (95% confidence interval). C) Quantification of polarization delays. Time was measured from the end of the first cytokinesis after the onset of pheromone treatment. Cells were considered polarized when a stable focused Spa2 cap was formed. Only those cells whose progenitors had completed cytokinesis during the first three hours after the start of pheromone treatment were considered. At least 52 cells (MP 384) were used to obtain the averages shown for each point. D and E) Cells arrested in G1 by G1 cyclin depletion were placed in exponential dilution chambers together with wild type cycling cells (using a fluorescent cell wall marker to distinguish the two strains). Experiments and measures are similar to those shown in B) and C). The transition to shmooing occurs at exactly the same concentration for G1 arrested and cycling cells (G1 arrested cells that do not shmoo grow isotropically, forming large spherical cells). Because Cln-depleted cannot leave G1 and cells arrested in G1 never polarize except when they form a shmoo, we do not show the delay before rebudding for wild type cells. The delay before shmooing is similar for cycling cells and for G1-arrested cells. In graph D) each point corresponds to an average value computed over 66 cells (MP 384 and MP 1333) and in graph E) over 52 cells (MP 384 and MP 1333).