Modular organization of the yeast PCD protein interaction network. (A) Visualization of the modularized PCD protein interaction network. Five modules with distinct functions constitute this network: Signaling (red), Transport (brown), Endocytosis (blue), Exocytosis (green), and Mitosis Exit regulation (gray). The different node shapes represent different universal roles as indicated. Cdc42 protein is used as an example to explain the method for reduction of the local connectivity of each node to the global connectivity to the modules with assigned kinetic parameters. (B) Description of the interactions (colored lines) of Cdc42 with proteins in different modules (large circles). (C) Mathematical representation of PCD protein interaction network and dynamics. The global connectivity of Cdc42 is represented by a 5D integer vector composed of the number of Cdc42’s linkages with each of the five modules. Each module is also assigned a characteristic time constant, τ_i (τ_1–5 for Signaling, Endocytosis, Transport, Mitotic Exit, and Exocytosis, respectively).

Experimental measurements of protein dynamics using iFRAP. (A–C) Images from iFRAP experiments and sample curves for proteins GFP-Rho3, GFP-Rho1, and Pea2-GFP. Red lines encircle bleached areas, and yellow lines encircle areas of fluorescence measurements. Red arrows indicate the time of iFRAP bleaching. The raw fluorescence measurements were subjected to monoexponential curve fitting (solid red curves). (Scale bar: 2 μm.) (D) Dynamics (measured as iFRAP t_1/2) of 11 proteins in the Signaling module. Boxes show the range of 1 SEM from the mean, represented by small squares. Vertical bars show the range of 1 SD from the mean. The horizontal line in the boxes represents the median. (E) Dynamics (measured as t_1/2) of 18 proteins in the Transport module represented as described in D. Note that the vertical time scale (0–50 s) for the 15 proteins on the left is different from that (0–180 s) for the 3 proteins on the right.

Correlation of measured vs. model-predicted residence times and training set analysis. (A) Correlation of mean measured residence times with the values predicted by the model, utilizing the characteristic module times (τ_i) calculated using the 10-component training set, for the proteins in the Signaling and Transport modules (black dots) and 2 proteins in the Mitotic Exit module (blue dots). The three outliers (Pea2, Rho1, and Lrg1) are represented by brown dots. (Inset) Correlation for the training set components (green dots). (B) Performance of qualified training sets randomly drawn (black dots) from the measured t_1/2 values of 29 proteins in Signaling and Transport modules in predicting the residence times for the 19 remaining proteins in these modules. The y axis represents the errors (as square root of error mean square) obtained by the least-squares multivariate regression method using each training set (Materials and Methods). The x axis indicates the correlation coefficient between the measured t_1/2 values and predicted residence values. The red point indicates the performance of our chosen training set. (C) Same analysis and representation as in B, except for predicting residence times for 21 proteins (19 as in B plus the 2 proteins in the Mitotic Exit module).

Effects of the scaffold protein Pea2 on the dynamics of its interacting proteins. (A) Local hierarchical organization of protein dynamics descending from Pea2 (green). It can be observed that proteins (red) more closely interacting with Pea2, generally exhibit longer t_1/2s than those (blue) further away from Pea2 in the hierarchy (the only exception is Yor304c-a). Shown are those components for which residence times were determined by iFRAP analysis. (B) Effects of pea2Δ on the dynamics of a subset of proteins in the hierarchy depicted in A examined by iFRAP. Shown are % changes of t_1/2 in pea2Δ compared with those in the WT. Red bars represent proteins in the higher hierarchy that directly interact with Pea2, and blue bars represent those in the lower hierarchy shown in A.

Effects of Pea2 on polarization kinetics and the rate of polarized growth. (A) Observation of GFP-Rho1 polarization in WT (black) and pea2Δ cells (green) by time-lapse movies (Movies S4 and S5). The plots show representative time-dependent changes in the average fluorescence intensity of GFP-Rho1 in the PCD, normalized against the average intensity in the whole cell. (B) Quantification of the rates of Rho1 polarization in time-lapse movies as described in A. The polarization rate was defined as the slope at the inflection point of the sigmoid curve used for fitting. Statistical representation is the same as described in Fig. 2D. (C) Example cell images for cell wall growth during budding in WT and pea2Δ cells by fluorescent Con A staining. Regions staining only with Texas Red Con A but not FITC Con A (red-only zones in merged images) represent a recent area of cell wall growth. (Scale bar: 2 μm.) (D) Quantification of cell wall growth during budding in WT and pea2Δ cells by fluorescent Con A staining. Plots show the increase in the area of growth over time. Averages of measurements from 15 to 25 cells per time point per strain are shown. Error bars reflect SEM.