morphology, or abundance of actin patches (data not shown), suggesting that Myo3 selectively promotes the transport of AP mRNAs to actin patches, their retention at actin patches, or both. Actin Patch mRNA Localization Patterns are Distinct from Those of ASH1 and ARG3 mRNAs Expression of the MS2-GFP protein in the cytoplasm of AP- MS2 cells had the incidental benefit of demarcating several cytological features; the GFP signal is excluded from the vacuole [8] and slightly enriched in the nucleus. Since the cell cycle phase of S. cerevisiae cells can be inferred from bud size, nuclear positioning, and the asymmetric localization of actin patches, we carried out a careful examination of AP mRNA localization across all stages of the cell cycle and at discrete cellular locations (Table S3). We used Fisher’s exact test with the Benjamini-Hochberg multiple hypothesis correction [102] to evaluate the significance of the observed differences in localization patterns between pairs of RNAs. The results are schematized in Figure 3B. For example, in the first ideogram of Figure 3B, the cumulative localization of BBC1:MS2 at cellular structures across all cell cycle stages is compared to the corresponding results for the ARG3:MS2 localization profile (comparisons for individual stages of the cell cycle are available in Table S3 and Figure S1). Discrete mRNA localization patterns in each comparison were color-coded to reflect significant relative over- or under-representation, with differences at an FDR of 161022 represented by light red or blue, respectively, ranging to differences at an FDR of 1610216, represented by dark red or blue, respectively. When an mRNA’s observed localization did not fall into any of the specific categories (presumptive bud, bud, bud neck, cell cortex, actin patch, nucleus, or vacuole), it was assigned the general designation of ‘‘cyto- plasm’’. It should be noted that this default category could encompass a diverse set of specific cellular structures that are not discernible in these images. Compared to the control mRNA, ARG3, ASH1 and AP mRNAs were enriched at the actin patch; BBC1, LAS17, and ASH1, but not VRP1, were also enriched at the cell cortex (the first four ideograms of Figure 3B). The apparent enrichment of VRP1 at the nucleus could reflect localization of nascent transcripts at the nuclear periphery [102], mRNAs in transit or docked at the nuclear pore complex, or mRNAs in association with perinuclear endoplasmic reticulum (ER), which is semi-continuous with the nuclear envelope. In addition, some differences in localization were seen among the AP mRNAs. BBC1 mRNAs, for example, were enriched relative to ARG3 mRNAs at the presumptive bud (Figure 3B, first ideogram), a site where actin patches cluster during bud formation in the early S phase of the cell cycle [103,104,105]. In contrast, LAS17 mRNAs, but not BBC1 or VRP1 mRNAs, were enriched in the definitive bud. BBC1 mRNA localization to the actin patch and cell cortex was also more pronounced than that of VRP1 mRNA (Figure 3B, ideogram 9), while both LAS17 mRNAs and VRP1 mRNAs were more frequently localized to the nuclear compartment than BBC1 mRNAs. ASH1 mRNAs were preferen- tially localized to the bud and bud neck, consistent with earlier reports [6,7,8], when compared to either ARG3 (Figure 3B, ideogram 4) or the AP mRNAs (Figure 3B, ideograms 5–7). Thus, while ASH1 mRNA and the AP mRNAs localized to actin patches in a similar fraction of cells (Figure 3A), the specific sites in the cell where this interaction took place were quite different. Addition of latrunculin B dramatically decreased the frequency of BBC1 mRNA localization to the presumptive bud and actin patch (Figure 3B, ideogram 11), but not the overall frequency with which it localized to the cell cortex. More tellingly, deletion of Actin Patch mRNA Localization and Dynamics MYO3 results in a redistribution of BBC1 and LAS17 mRNAs from the presumptive bud, bud, cell cortex, and actin patches to the cytoplasm, indicating that Myo3 is critical for association of these AP mRNAs with actin patches and at particular sites within the cell. Surprisingly, deletion of MYO3 also resulted in a shift in ARG3 mRNA localization from the vacuole to the nucleus, the basis of which is a mystery. To investigate the mechanism of recruitment to actin patches, which are dynamic cortical structures [92,94,97], we visualized the LAS17:MS2 mRNP at the presumptive bud by two-color timelapse microscopy using FM4–64 to visualize sites of endocytosis (coincident with actin patches [106]). In an observation over more than four minutes (Figures 4A and 4B, Video S1), a LAS17:MS2 mRNP seen at the cluster of actively-endocytosing actin patches at the presumptive bud moved less than 500 nm during the timelapse movie (Figure 4B), strongly suggesting that it was confined or effectively anchored at the actin patch. Indeed, we observed many mRNAs residing at the presumptive bud site for extended periods lasting up to 10 minutes (data not shown). We frequently observed cortical anchoring interspersed with rapid, erratic movement of the mRNA particle consistent with diffusion, as demonstrated by the LAS17:MS2 mRNP in the M phase cell in Figures 4C and 4D (see also Video S2). Examination of the particle trajectory (Figure 4D) shown in the montage in 4C reveals that the mRNP can move along the cell cortex over a distance of more than 2 mm between sites of prolonged residency. The apparent short-term, 2D diffusion coefficient (Figure 4E) of this mRNP did not change between 10 to 20 s; it then appeared to be briefly confined from 20 to 55 s, rapidly diffusive between approximately 55 to 70 s, then ‘‘recaptured’’ and relatively immobile from 100 to 120 s, suggesting the possibility of a diffusion and capture mechanism for localization. Tracking Single BBC1 mRNPs with High Spatial Precision in Three Dimensions Preliminary studies (Figure 4) revealing diverse dynamic behaviors of mRNPs at different stages of the cell cycle and within single particle trajectories convinced us that endpoint analyses of localization and analyses of the 2D trajectories with the spatial precision of a standard epifluorescence microscope would provide an inadequate picture of the 3D dynamics of mRNPs. Indeed, standard widefield fluorescence microscopy has localiza- tion precision in the axial dimension (500–800 nm) that is several times larger than the localization precision needed to accurately measure fine-scale dynamics, such as localized confinement. For example, our previous work with the DH-PSF system showed that mRNPs exhibiting movement as large as 1.5 mm in z could be erroneously described as stationary or confined based on standard 2D widefield fluorescence microscope imaging [107]. To over- come these limitations, we used a double-helix point spread function (DH-PSF) microscope to characterize mRNP dynamics at high temporal and spatial resolution in 3D; the system and method are more thoroughly described elsewhere [107,108]. Briefly, the DH-PSF microscope uses a spatial light modulator with a phase mask and Fourier plane processing [109,110] to convert a single, diffraction-limited fluorescent spot, in our case a BBC1 mRNP, into two spots that are detected in the DH-PSF imaging plane by the electron multiplying charge coupled device (EMCCD) camera (Figure 5A). Translation of the single particle along the z-axis is reflected by rotation of the two spots about their midpoint, effectively carving out a double helix within a 2 mm depth of field, thus allowing one to infer the position of a single particle in z with high spatial precision based upon the angle between the two spots; x and y are extracted from the midpoint between the two spots. In PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31912