confinement and simulations indicates that the BBC1 confinement events are not false positives and cannot be solely described by Brownian dynamics. It should be noted that these non-Brownian confinements were also observed in ARG3 [107], and might be explained by interactions with structures in the cellular mRNP microenvironment. For the confinement size distributions, the mean confinement size in each case was 0.2160.09 mm, 0.2160.07 mm, and 0.2960.17 mm for BBC1 untreated, BBC1 myo3D, and BBC1+latB, respectively (Figure 6I–K, Dataset S3). The confinement size distribution predicted for a particle diffusing in a homogeneous medium with the same apparent diffusion coefficient (D) as BBC1 (false positives from the Brownian mimic) were considerably larger and more varied with a mean value of 0.4560.4 mm and the distributions for the three experimental cases were all significantly different from the simulated distribution (BBC1, P,10211; BBC 1 myo3D, P,10212; BBC1+latB, P<0; Table S6). This indicates that BBC1 binding events restrict the particle to a smaller volume than expected for Brownian fluctuations. The deletion of MYO3 did not have a significant effect on the observed confinement size (P = 0.87), although the addition of latrunculin B did result in significantly larger confinement size when compared to both BBC1 untreated and BBC1 myo3D (P = 0.001 for both). These results suggest that the effects of Myo3 on BBC1 mRNP local confinement size are minimal and that when BBC1 is not confined in an actin dependent manner (e.g. bound to an actin patch), then it is diffusing with intermittent short time interactions, a phenomenon also observed in ARG3 [107]. However, introduction of latrunculin B increases the volume in which the BBC1 mRNP is confined, suggesting that actin cables or patches play a significant role in restricting the local dynamics of the mRNPs. Discussion We used a systematic approach to identify the mRNAs associated with the cytoskeletal motor proteins of S. cerevisiae. We found consistent association of distinct functionally related sets of mRNAs with 4 of the 5 known yeast myosins, Myo1, Myo2, Myo3, and Myo4, and 2 of the kinesin-like proteins, Kip3 and Kar3. In total, approximately 1,000 mRNAs are associated with cytoskeletal motor proteins in S. cerevisiae, demonstrating that mRNA association with motors is pervasive and may be an important means of generating widespread mRNA asymmetries. We also sought to determine the mechanism by which motor- dependent mRNA localization might be occurring. We chose to focus further investigation on the interaction of mRNAs encoding key regulators of actin patch dynamics with a type I myosin, Myo3. We tagged the endogenous LAS17, VRP1, and BBC1 loci with 12 MS2 hairpin aptamers which, when expressed in conjunction with the EGFP-tagged MS2 hairpin binding protein, allowed us to visualize mRNP localization in live cells. Using an mCherry-tagged Sac6 protein to mark actin patches, we confirmed that these AP mRNAs localized to actin patches significantly more often than did a control mRNA, ARG3, and at a frequency comparable to ASH1 (Figure 2 and Figure 3). The AP mRNAs associated with actin patches at sites distinct from ASH1, with ASH1 exhibiting a stronger preference for localization to actin patches in the bud or bud neck (Figure 3). The differential localization of AP and ASH1 mRNAs to actin patches in distinct parts of the cell suggests both that these mRNAs use different mechanisms for delivery or anchoring to actin patches and that the actin patches at different cellular locations may be molecularly distinct. Both Myo3 and F-actin were critical for maintaining AP mRNA localization at actin patches, suggesting that Myo3 is Actin Patch mRNA Localization and Dynamics important for either delivery of the AP mRNA to actin patches or anchoring of the transcript at these sites, or both. Dynamics of a Myo3-Associated Actin Patch mRNA, BBC1 To complement our study of the static localization of mRNAs in live yeast cells and to further investigate the mechanism of AP mRNA localization to actin patches, we combined low resolution, 2D imaging of mRNP dynamics on long time scales with high temporal resolution, 3D super-localization (DH-PSF) microscopy of single particle dynamics on short time scales. 2D imaging results suggested that AP mRNAs associate with sites of actin patch enrichment for extended periods of time (Figure 4), alternating with periods of diffusion, consistent with a diffusion and capture model for localization of AP mRNAs to the actin patch. Because critical features of the dynamic behavior of mRNAs occur in a spatial and temporal scale much finer than can be resolved by conventional epifluorescence microscopy, we used a 3D super-localization microscopy method [107,108,114] that provides a spatial precision of 25 nm in x and y and 50 nm in z at a temporal resolution of 50 ms to observe several hundred BBC1 mRNA trajectories. Neither depolymerization of actin nor deletion of MYO3 affected the average 3D diffusion coefficient of BBC1 particles, suggesting that diffusive movement of the particle is not greatly influenced by either of these factors. The distribution of BBC1 diffusion coefficients was significantly different from that of the control mRNA, ARG3 (,D. = 0.03960.02 mm2/s, P = 0.022; [107]), demonstrating that distinct mRNPs have characteristic dynamic properties, perhaps dependent upon mRNP makeup and local cellular environment. Deletion of MYO3 had little effect on the apparent confinement time and volume of the BBC1 particle (Figure 6). The apparent confinement times of BBC1 mRNAs in either wildtype or MYO3 cells were significantly greater than expected under a model of unconstrained diffusion, indicating that the BBC1 mRNPs episodically interact with cellular structures that restrict or impede their movement. Although the results were not significant by the Kolmogorov-Smirnov or Mann-Whitney tests with the size of the current dataset, examination of the cumulative distribution function (CDF) plot in Figure 6H demonstrates that deletion of MYO3 and addition of latrunculin B cause a shift to shorter confinement times, approaching the values obtained in the simulation. This result is consistent with decreased AP mRNA localization to actin patches in MYO3 cells (Figure 3) and provides further evidence of a potentially important role for Myo3 and F-actin in the confinement of BBC1 mRNPs. Depolymerization of F-actin by the addition of latrunculin B decreased the association of AP mRNAs with actin patches (Figure 3). A significant shift in the distribution of confinement sizes (Figure 6 I–L) indicates that actin plays a critical role in limiting the confinement radius of BBC1. Model for BBC1 mRNP Recruitment to Actin Patches There are several models that could be considered for the apparent roles that Myo3 and F-actin play in the localization and dynamics of the BBC1 mRNP. First, as Myo3 is an actomyosin motor, one a priori model would posit that AP mRNAs undergo Myo3-associated directed transport along actin cables to the actin patches in a manner similar to Myo4-mediated ASH1 transport to the bud tip [6,9,115]. Myo4 is a type V, non-processive motor as a monomer, meaning that there is a low probability that a single Myo4 motor will stay associated with an actin filament through many enzymatic cycles. However, Myo4 becomes processive upon self-assembly into a multi-motor oligomer, suggesting that multiple Myo4 molecules bound to the ASH1 mRNA cargo facilitate its localization to the bud tip [116]. In contrast, Myo3 is a member of PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e31912