Y, that a random walk would stay within a circle of that radius. For each trajectory, the average diffusion coefficient and localization precision were measured and Brownian simulations were run to determine the 90%, 95%, and 98% thresholds for confinement. To calculate log(Y), the trajectory was segmented into sub-trajectories that were 30 time points long. For each sub-trajectory, a maximum displacement R is measured from which log(Y) can be calculated in each window. If the calculated log(Y) is less than the threshold than the particle is denoted as confined. The confinement radius is the average maximum displacement, R, during the confined time window and the confinement time is the time from when the particle first enters a confined state until the particle exits the confined state. Supporting Information Dataset S1 Significance analysis of microarray results for each immunoaffinity purification approach for the actomyosins, Las17, and Vrp1. (TXT) Dataset S2 Significance analysis of microarray results for each immunoaffinity purification approach for the kinesin-like proteins and dynein. (TXT) Dataset S3 Diffusion coefficients and confinement times and sizes for BBC1 at different confidence thresholds. (XLS) Figure S1 Quantification of mRNA localization at cellular loci at all stages of the cell cycle. Significant differences in localization between mRNAs of interest (columns) at cellular locations (rows) are indicated by color. Cellular locations in cells at different stages of the cell cycle are schematized on the left. Red or blue indicates a –log false discovery rate (FDR) of 2 (light color) to 16 (saturated, dark color), with red colors indicating significant over-representation of mRNA 1 in each pair and blue colors indicating significant over-representation of mRNA 2. Significance was measured by Fisher’s exact test. (TIF) Figure S2 Example trajectory of unbiased diffusion of mRNP from model. An example trajectory for the unbiased diffusion model is displayed along with the outline of a theoretical cell and the total time elapsed before the randomly diffusing mRNP reaches the actin patch. The trajectory is encoded by color with the beginning of the trajectory indicated by red and the end in purple. (TIF) Figure S3 Example Trajectory of Biased Diffusion of mRNP from Model. An example trajectory for the biased diffusion model, in which the non-processive motor-associated mRNP takes 8 nm steps toward the actin patch once per second, on average, is displayed along with the outline of a theoretical cell and the total time elapsed before the mRNP reaches the actin patch. The trajectory is encoded by color with the beginning of the trajectory indicated by red and the end in purple. (TIF) Video S1 LAS17:MS2 mRNP dynamics in a cell incubat- ed with FM4–64 to reveal endocytic actin patches. Movie of LAS17:MS2 mRNP displayed in Figure 4A. Time is displayed in seconds and the scale bar represents 2 mm. (AVI) Actin Patch mRNA Localization and Dynamics Video S2 LAS17:MS2 mRNP dynamics. Movie of LAS17:MS2 mRNP displayed in Figure 4C. Time is displayed in seconds and the scale bar represents 2 mm. (AVI) Table S1 S. cerevisiae strains used in this study. Yeast strains acquired or constructed for this study. Strains JCY18 [129] and GFP strains, including the wildtype parent JCY93 [45], have previously been published. (XLS) Table S2 Percentile rank of mRNAs encoding actin patch associated proteins in IPs of motor proteins. The average log2 Cy5/Cy3 ratio of all mRNAs from IPs of Myo1, Myo2, Myo3, Myo4, Myo5, Las17, and Vrp1 with GFP or 9Myc tags and with the addition of latrunculin B (‘‘latB’’) or upon formaldehyde crosslinking (‘‘xlink’’) were calculated and ranked. The percentile ranks of all mRNAs from the GO category ‘‘actin cortical patch’’ (GO:0030479) in S. cerevisiae are displayed. (XLS) Table S3 Counts of mRNA localization at cellular loci at all stages of the cell cycle. Counts of mRNA localization for ARG3:MS2, ASH1:MS2, BBC1:MS2, LAS17:MS2, and VRP1:MS2 with and without cellular perturbations at cellular locations during all stages of the cell cycle. (XLS) Table S4 BBC1 diffusion coefficient statistical tests. Kolmogorov-Smirnov (K-S) and Mann-Whitney (M-W) statistical tests were performed for the distribution of diffusion coefficients (Dataset S3) of BBC1 untreated, MYO3 (‘‘BBC1 myo3D’’), and cells treated with latrunculin B (BBC1+latB). The two-tailed M-W p-value is reported. (XLS) Table S5 BBC1 confinement time statistical tests. K-S and M-W statistics at the 90%, 95%, and 98% confidence thresholds are displayed as in Table S3. (XLS) Table S6 BBC1 confinement size statistical tests. K-S and M-W statistics at the 90%, 95%, and 98% confidence thresholds are displayed as in Table S3. (XLS) Table S7 Oligonucleotide primers used in this study. Oligonucleotides indicated by an asterisk (*) for generating gene disruptions were published in Winzeler et al. [132]. (XLS) Text S1 (DOC) Description of modeling. Text S2 Code written in R for single particle diffusion modeling in S. cerevisiae. (DOC) Acknowledgments We are grateful to Dr. Roger Tsien, Dr. Jeffrey Gerst, Dr. Pamela Silver, Dr. Erin O’Shea, and the Yeast Resource Center for supplying yeast strains and plasmids. We also appreciate the contribution of Nikoleta Tsvetanova, Deyra Rodriguez, Jason Myers, and Daniel Hogan for early help in motor protein purifications; Dr. Aaron Straight, Dr. Mark Krasnow, Patrick Bogard, and Ben Moree for microscope use and microscopy advice; Colin Fuller, Mandi Hartman, and Christopher Toret for helpful conversations; and members of the Brown and Moerner labs for advice and discussions. We would also like to thank Raymond Chen, Dustin Hite, Ariel Jaimovich, Liana Lareau, and Deyra Rodriguez for comments on the manuscript. PLoS ONE | www.plosone.org 17 February 2012 | Volume 7 | Issue 2 | e31912