regulating mRNAs. The weak or absent association of Myo5, Las17, and Vrp1 with AP mRNAs, as well as the presence of EDTA in our purification buffers (see Materials and Methods), which would disrupt any polysomal mRNAs that might interact with Myo3 indirectly through actin patch-localized ribosomes, suggests that Myo3’s association with key AP mRNAs is specific. Third, there is precedent for the localization of b-actin mRNA and mRNAs encoding the seven Arp2/3 components to sites of actin branching and cytoskeletal rearrangement in mammalian cells [20,21,22,23,24,25]. Because actin patches are focal points of cytoskeletal polarity in S. cerevisiae, our result suggests that Myo3 association with AP mRNAs may reflect a common mechanism for localization of mRNAs encoding actin cytoskeleton polariza- tion via association with the actin cytoskeleton itself. Myo3-Associated mRNAs Localize to Actin Patches To test the hypothesis that the Myo3-associated AP mRNAs would localize to actin patches, we used a modified MS2-tagging system that allows expression of the mRNA from its native promoter and preserves the 39 UTR sequence [96]. We integrated sequences encoding a series of twelve MS2 coat protein binding RNA hairpins between the respective termination codons of BBC1, LAS17, and VRP1 and their respective 39-UTR sequences in separate strains (Figure 2A and Materials and Methods). In each of these strains we expressed an MS2 coat protein (MS2-CP) lacking a nuclear localization sequence and fused to three tandem copies of EGFP under the control of a methionine-inducible promoter [8,96]. Upon induction, the MS2-CP-3xEGFP protein accumu- lates in the cytoplasm, but is selectively enriched at the site of the MS2 hairpin-tagged mRNAs, allowing for visualization of the corresponding mRNPs as distinct, bright foci [96]. Induction of the MS2-CP-3xGFP in a control strain lacking any MS2 hairpin RNAs led to bright foci in only 0.01% of cells (data not shown) and Actin Patch mRNA Localization and Dynamics their morphologies were distinct from those representing bona fide mRNP complexes. AP-MS2 mRNA haploid strains were mated to a strain in which the Sac6 fimbrin, a marker of actin patches [79,81,97], was genetically tagged at its C-terminus with the mCherry protein (Figure 2A). As displayed in Figure 2B, the AP mRNA GFP signal of the LAS17, BBC1, and VRP1 mRNPs co-localizes with Sac6 mCherry signal in images taken from live cells, indicating that the AP mRNPs localize to actin patches. In order to quantify the coincidence of AP mRNPs at actin patches, we examined thousands of two-color images of live cells harboring AP mRNPs or two control mRNAs, ARG3 and ASH1. The ARG3 mRNA, which encodes ornithine carbamoyltransferase in S. cerevisiae, was chosen because it was not bound by Myo3 in the presence of formaldehyde or latrunculin B (Dataset S1), is expressed at just 1– 2 mRNA copies per cell, similar to the AP mRNAs [98,99], and because it encodes a cytoplasmic enzyme that is not known to exhibit asymmetric localization [45,100,101]. ASH1 was chosen as a putative positive control; while ASH1 mRNA was not found associated with Myo3, previous studies have indicated that ASH1 may anchor at cortical actin in the bud tip [7,8]. Deletion of MYO3 and Disruption of Actin Impair Localization of Actin Patch mRNAs to Actin Patches As shown in Figure 3A, we observed significant differences among VRP1, LAS17, BBC1, and ASH1 in the proportion of mRNAs localized to actin patches (19%, 29%, 35%, and 29%, respectively) when compared to the control mRNA, ARG3 (8%). Pairwise differences in the proportion between ARG3 and each of VRP1, LAS17, BBC1, and ASH1 were each significant by Fisher’s exact test (P = 3.761024, P = 3.961028, P = 2.2610212, and P = 1.961028, respectively; Table S3). Addition of latruculin B, which destabilizes F-actin, also led to a significant decrease in the Figure 2. Actin patch nucleator mRNAs are localized to actin patches. (A) Schematic of fluorescent strain construction for visualization of mRNA particles and actin patches in live cells. The sequence for 12 MS2 hairpins was integrated between the coding region of the gene of interest (e.g. BBC1) and the 39 UTR at the native genomic locus. Cells with the gene:MS2 construct also harbored a plasmid allowing inducible expression of the MS2 hairpin-binding coat protein (CP) fused to three tandem copies of enhanced green fluorescent protein (3xGFP), allowing visualization of discrete mRNA particles in live cells. Haploid strains harboring the MS2 constructs were mated to haploid strains harboring an integrated mCherry C- terminal fusion of the S. cerevisiae fimbrin protein, Sac6p. (B) Visualization of MS2-CP-3xGFP tagged mRNAs (left panel) and Sac6p:mCherry (middle panel) in diploid cells. Areas of interest in the left panel, highlighted by the yellow bounding box, are shown in the right panel. mRNAs of interest are highlighted by a white arrow. Scale bars are 2 mm. doi:10.1371/journal.pone.0031912.g002 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31912