to visualize the dynamics of the mRNP in live cells [9]. Subsequent genome-wide studies have indicated that more than 20 transcripts associate with Myo4 and translocate from the mother cell to the budding daughter cell tip, where they, along with ASH1, are believed to be anchored in an actin-dependent manner [6,7,8,13,18,19]. Actin-dependent mRNA localization appears to be widespread [1,2,3]. One of the best-studied cases is the mRNA encoding b- actin itself. b-actin mRNA localizes to lamellapodia at the leading edge of crawling chick and mammalian fibroblasts [20,21,22]; its localization is required for the enhancement of cytoskeletal polarization, cell motility and directionality in crawling [23,24]. Localization of b-actin mRNA correlates with locally elevated levels of b-actin protein, supporting the model that transcript localization facilitates localized translation of b-actin [23]. As actin is highly dynamic, localized production of the b-actin monomer would increase the local concentration of the b-actin pool to facilitate the rapid polymerization necessary for cytoskeletal rearrangement and cell movement. Further studies have indicated that the seven mRNAs that encode the Arp2/3 subunits, which are necessary for actin branching, are also localized to the leading edge of fibroblasts [25]. From the studies of b-actin and Arp2/3 mRNA localization, an interesting model has emerged in which cells reinforce polarization by transporting mRNAs encoding proteins that modify cytoskeletal polarization along the cytoskel- eton itself [23,25]. Microtubules and the associated dynein and kinesin motor proteins are also implicated in dynamic mRNA localization in higher organisms [1,2,3]. For example, both bicoid and gurken mRNAs have been shown to undergo dynein-dependent localiza- tion in Drosophila oocytes [26,27,28,29]. Interestingly, the dynein motor that facilitates movement of the gurken mRNA to the anterior of the oocyte serves as an anchor for the transcript once it is properly localized, indicating that motor proteins may play multiple roles in dynamic mRNA localization [30,31]. Several mRNAs have been found to have kinesin-dependent localization, including Vg1 mRNA in Xenopus oocytes [32,33] and oskar mRNA in Drosophila oocytes [34], CaMKIIa in mammalian neurons [35], and MBP in oligodendrocytes [36]. Vg1 mRNA, which encodes a member of the transforming growth factor b family, is localized to the vegetal pole of Xenopus laevis oocytes [37]; its localization depends on two different kinesins in a portion of the oocyte with mixed microtubule orientations [38]. Studies of oskar mRNA dynamics in live Drosophila oocytes, where Oskar localization to the posterior pole is important for body axis patterning [3], have revealed a similarly complex localization mechanism. Quantification of oskar dynamics in live cells has suggested that oskar mRNA is able to move in a kinesin-dependent manner in all directions along a mixed-polarization microtubule cytoskeleton with a weak overall bias towards the posterior [39]. As evidenced in the studies of Vg1, oskar, and ASH1, careful measurement of mRNP dynamics has been critical for revealing the mechanisms that impart asymmetric sorting. It is clear that various proteins and signaling events alter the composition and presumably the dynamics of the ASH1 mRNP between its birth in the nucleus and its ultimate localization in the cell. Recent studies of ASH1 localization have revealed that the She2 protein is recruited to the mRNA co-transcriptionally and is believed to accompany the mRNA into the cytoplasm, where it encounters Myo4 for eventual translocation to the bud tip [40]. Another RBP, Puf6, serves as a translational repressor, believed to associate with the mRNA until the ASH1 transcript is released from repression at the bud tip [41,42]. Further studies have demonstrated that paralogous S. cerevisiae ribosomal proteins, previously thought to be Actin Patch mRNA Localization and Dynamics functionally interchangeable, differentially affect the ability of ASH1 to be properly localized [43]. This finding suggests the intriguing possibility that different classes of ribosomes themselves may differentially associate with specific mRNAs, providing an additional mechanism for regulation of mRNA localization. While most studies of mRNA dynamics in S. cerevisiae have focused on ASH1 and other transcripts associated with Myo4, we hypothesized that other cytoskeletal motor proteins might have roles in mRNA localization in yeast. In order to test this hypothesis, we immunopurified the five actomyosin motors, Myo1, Myo2, Myo3, Myo4, and Myo5; the kinesin-like proteins Kar3, Kip1, Kip2, Kip3, and Smy1; and the dynein, Dyn1, from S. cerevisiae and profiled associated RNAs using DNA microarrays. This analysis established that six of the eleven cytoskeletal motor proteins associate with functionally specific sets of mRNAs, indicating that asymmetric localization of mRNAs via association with motors is widespread. Among the new mRNA interactions identified is the association of Myo3 with mRNAs encoding key regulators of endocytosis and actin patch dynamics. We used conventional fluorescence microscopy and a microscope exhibiting a double-helix point spread function (DH-PSF) for 3D super- localization microscopy of MS2-GFP-tagged mRNAs to quantita- tively profile the localization and dynamics of a Myo3-associated actin patch mRNA at high temporal and spatial precision in three dimensions. Tracking of single actin patch-localized mRNPs with an average spatial precision of 25 nm in x and y and 50 nm in z allowed us to derive 3D diffusion coefficients and measures of confinement, as well as quantitative measurements of the contribution of Myo3 and filamentous actin (F-actin) to actin patch mRNA dynamics, providing insight into the mechanism of actin patch (AP) mRNA localization to actin patches in live cells. Results Immunopurification of Cytoskeletal Motor Proteins To enable efficient immunopurification (IP) of cytoskeletal motor proteins in S. cerevisiae, we constructed strains in which nine Myc epitopes were fused to the C-terminus of each of six cytoskeletal motor proteins by knock-in at the endogenous loci ([44]; Materials and Methods). For some studies, we used strains in which a C-terminal enhanced green fluorescent protein (EGFP) was fused to selected motor proteins ([45]; Table S1). We used formaldehyde crosslinking to stabilize associations between motors proteins and interacting RNAs before IP. Yeast cells growing exponentially in rich medium were treated with formaldehyde, lysed and sonicated, then incubated with magnetic beads coupled to monoclonal antibodies against either 9Myc or GFP (Figure 1A) to isolate the tagged motor proteins along with any associated RNAs. After IP, the formaldehyde crosslinks were reversed and the enriched RNAs and RNAs purified from the corresponding total lysate were amplified and labeled respectively with Cy5 and Cy3, then jointly hybridized to custom-made, S. cerevisiae oligonucleotide microarrays [46]. Several controls for the IPs were performed. Out of concern that formaldehyde might stabilize spurious association of mRNA with the motor proteins, we attempted alternative purification approaches that did not involve crosslinking. While soluble motor proteins could be immunopurified in the absence of formaldehyde, their specific association with mRNAs was less consistent in the absence of crosslinking, and much of the motor protein was found to partition to the insoluble fraction (data not shown). The addition of a low concentration of latrunculin B (2 mg/ml) to live cells to partially solubilize the actin cytoskeleton [47] allowed for successful IP of the motor proteins and associated mRNAs without PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31912