Figure 5. Experimental design of synthetic PIP₃ polarization networks.

(A) Three regulatory links were introduced into a yeast strain by expressing all three synthetic fusion proteins from our toolkit. Several cells in one field of view show PIP₃ polarization with this most robust combination of polarization motifs (top right). In most cases, cells were treated with 5μg/ml Latrunculin A (LatA) before imaging to prevent budding. When omitting LatA treatment, PIP₃ (green) polarizes away from the bud site (phase), where Cdc42* is concentrated. (B) Timecourse of PIP₃ polarization demonstrates that they are dynamic but can persist for over tens of minutes (10 minute intervals between images). (C) Histograms of polarity scores (PS). Positive feedback alone (top) produces a small number of weak poles. The three-motif (bottom) combination produces significantly larger numbers of strong poles. Example images of a range of polarity scores are shown. N indicates the number of individual cells analyzed with each network topology. Maximum polarity score for each cell through observation is shown. See also Figures S5 and S6. (D) A three-dimensional rendering of a cell with polarized PIP₃ (See also Movies S1 and S2).