(62 nm). Because of drift in the microscope, either resulting from the sample or the microscope itself, it is not possible on the current setup to obtain high spatial precision (,50 nm) with an observation time of more than approximately 2–3 minutes. Based on the low-resolution two-dimensional work described above, it was clear that mRNPs bind to actin patches for periods exceeding four minutes; if these events are randomly observed in the DH- PSF studies window, most will be classified as stationary trajectories. In untreated BBC1:MS2 cells, 24 of 122 trajectories were stationary. In BBC1:MS2 myo3D samples, only 10 of 117 trajectories were stationary (P = 0.016), indicating that MYO3 is important for BBC1 mRNP anchoring. In latrunculin B treated cells, 12 of 104 trajectories were stationary (P = 0.10). The difference in stationary events between myo3D and the latrunculin B treated cells was not significant (P = 0.505), suggesting that both perturbations similarly decrease the number of BBC1 mRNP stationary events. By combining the number of stationary events under both perturbations (22 of 221 trajectories), as both actin and Myo3 may be independent contributors to anchoring, and comparing to the untreated BBC1:MS2 cells, we conclude that the Myo3-actin interaction is important for BBC1 mRNP anchoring (P = 0.013) as the BBC1 mRNP is far less likely to be stationary in cells lacking MYO3 or treated with latrunculin B. BBC1 mRNPs were more frequently diffusive in latrunculin B treated cells (70.7% versus 63.4%; Table 1) and when MYO3 was deleted (78.2% versus 63.4%; Table 1). Indeed, in myo3D cells the distribution of dynamic behaviors observed (stationary, diffusive, and confined) approached that of the control, ARG3, suggesting that some BBC1-specific confinement behavior is MYO3 depen- dent. BBC1 mRNPs Do Not Exhibit Directed, Processive Motion Although the dynamics of Myo3 motors in S. cerevisiae are still poorly understood, the association of a myosin motor protein with an mRNA might suggest directed or active transport of the mRNA molecules. In our previous work, a modified three-dimensional version of the speed correlation index (SCI) was used to identify directed motion [107,111]. Surprisingly, ARG3, which was not found to significantly associate with any motor proteins, displayed 40 events with long periods (.1 s) of actin-dependent directed motion in 267 trajectories [107]. When the SCI tests are applied to BBC1 trajectories, the long directed movements observed for ARG3 are absent (data not shown). The average ‘‘directed’’ trajectory for a BBC1 particle lasted less than 0.7 s, which is within the duration of short Brownian trajectories that would be erroneously described as ‘‘directed’’ by chance. Although Myo3 dynamics are not well characterized, type I myosins like Myo3 are generally thought to be non-processive motors (reviewed in [112]), making Myo3-dependent directed and processive movement over long time periods unlikely. Thus, we suspect that Myo3 may guide BBC1 mRNAs towards actin patches by non-processive steps that bias the diffusion of the mRNPs towards patches (vide infra). Deletion of MYO3 or Addition of Latrunculin B Do Not Affect Apparent BBC1 mRNP Diffusion Coefficients Analysis of the distribution of diffusion coefficients allows us to further quantitatively test the specific impact of Myo3 and F-actin on BBC1 mRNP dynamics. The average diffusion coefficient for BBC1 mRNA in latrunculin B treated or MYO3 cells is similar to that in unperturbed wildtype cells (D = 0.03060.03 mm2/s, 0.03460.04 mm2/s, and 0.02460.02 mm2/s, respectively; Figure 6A–C and Dataset S3); the distributions were not significantly different by the Kolmogorov-Smirnov (K-S) test or Actin Patch mRNA Localization and Dynamics the Mann-Whitney (M-W) test (Table S4). We observed BBC1 mRNPs with higher apparent diffusion coefficients in myo3D cells versus wildtype cells (Figure 6C vs. 6A); however, more observations will be required to establish the potential significance of this difference. The apparent diffusion rates of BBC1 mRNPs were consistently significantly slower that those of ARG3 mRNPs (0.039 mm2/s, P = 0.022; [107]); thus, different mRNPs have distinct and characteristic diffusion rates which we hypothesize to reflect differences in mRNP composition and subcellular micro- environment. The distribution of observed apparent diffusion coefficients was much broader than predicted from a model in which the particles and microenvironment are homogeneous (solid curve in Figure 6A–C and labeled ‘‘Simulation’’; Figure 6D). BBC1 mRNP Confinement on Short Time Scales is Non- Brownian is Affected by Addition of Latrunculin B As shown in Figure 5B, BBC1 mRNP exhibits confinement events interspersed with intervals consistent with diffusion, similar to our previous observations of ARG3 dynamics [107]. In order to determine whether Myo3 or F-actin contributed to the local confinement of BBC1 mRNPs in live cells, we used equation 1 [91,107,113] to quantitatively assess the confinement dynamics of all measured trajectories. log(Y)~0:301{4:286(Dtw=R2) 1 ð Þ To analyze the trajectories for confinement using equation 1, the trajectories are first split into sub-trajectories, each with a length, tw, of 30 time steps (0.45 s). The probability that the observed sub-trajectory reflects bona fide confinement or is explained by Brownian motion (i.e. false positive) is calculated as the log ofY. An MSD analysis is performed on the entire trajectory to obtain the apparent diffusion coefficient, D. By then measuring the maximum three-dimensional displacement in each sub- trajectory, R, we can now substitute the above values for tw, D, and R into equation 1 to obtain the probability, log(Y). For each trajectory, 1000 Brownian trajectories with the same length, localization precision, and diffusion coefficient were simulated to calculate a 95% confidence threshold (See Materials and Methods for a more detailed description). When the observed log(Y) surpasses (falls below) the threshold, then the subtrajectory is classified as confined, and when log(Y) is larger than the threshold then the subtrajectory cannot be significantly distinguished from Brownian motion. In Figure 6, these distributions are denoted as ‘‘Simulation’’. Results at the 95% confidence threshold are reported here and the test was also performed using 90% and 98% confidence intervals [Dataset S3]. The observed confinement times at the 95% confidence threshold were similar for all three experimental cases: 1.362.1 s, 0.8161.0 s, and 0.8161.2 s for BBC1:MS2 cells (untreated), cells for MYO3 (BBC1 myo3D), and cells treated with latrunculin B (BBC1++latB; Figure 6E–G and Dataset 3), respectively. Deletion of MYO3 or addition of latrunculin B did not affect the confinement time (P = 0.26 and P = 0.19, respectively; Table S5). However, BBC1 confinement periods in untreated, BBC1 myo3D, and BBC1+latB cells were much longer, on average, than the simulation (0.4260.3 s; P,0.0001, P,0.0001, and P = 0.004, respectively; Table S5). The significantly shorter confinement times in the simulation are due to short-lived Brownian fluctuations that represent false-positive classification as ‘‘confined,’’ which is similar to previous findings [113]. The significant difference between the observed apparent BBC1 PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31912