Actin Patch mRNA Localization and Dynamics Figure 7. Model of BBC1 mRNP association with actin patches. (A) BBC1 can be found in a diffusive state (large green circle) and a confined or stationary state (small green circle) in association with the endocytosing actin patch. Myo3 is represented by a blue rod and actin is colored in red. (B) Deletion of MYO3 (myo3D) leads to decreased association of BBC1 with the actin patch and a decrease in the proportion of BBC1 mRNPs in the stationary state (large downward arrow). (C) Disruption of F-actin via addition of latrunculin B causes a decrease in the proportion of BBC1 mRNPs at the actin patch (large downward arrow) and an increase in confinement size (mid-sized green circle). Average diffusion coefficients and confinement sizes are displayed. doi:10.1371/journal.pone.0031912.g007 the type I family of non-processive actomyosin motors, which are monomeric, have characteristically slow rates of ATP turnover and actin movement, and thus not expected to be capable of extended directed motion along actin filaments (reviewed in [112,117]). Indeed, we failed to detect significant sustained and directed motion of the BBC1 mRNP, even on short time scales. A second model for Myo3 and actin-dependent localization involves Myo3 serving solely as an anchor for AP mRNAs at the actin patch. In this model, randomly diffusing AP mRNAs would become trapped, or anchored, at actin patches in an actin- and Myo3-dependent manner, consistent with the decrease in AP mRNA localization to actin patches we observed upon deletion of MYO3 or treatment with latrunculin B (Figure 3 and 7). Indeed, an anchor-like role for a motor protein has been reported in which a dynein motor was shown to serve as an anchor for proper gurken localization in Drosophila [30,31]. A third model posits that Myo3 associated with the BBC1 mRNP facilitates a biased diffusion towards actin patches. In this model, the saltatory but consistently polarized steps of Myo3 on actin filaments that are themselves polarized toward the actin patches [118,119] results in a slight but consistent and cumulative bias in mRNP movement towards areas of actin nucleation such as the actin patch. In order to conceptually test this third model, we performed simulations to determine how long a freely diffusing mRNP with the apparent diffusion coefficient of BBC1 (0.025 mm2/s) would take, on average, to reach an arbitrary point on the cell surface representing the cluster of 23 spherical actin patches [81] in an S phase cell, each 140 nm in diameter, while being excluded from the nucleus and vacuole (see Text S1 and Text S2 and example trajectories in Figure S2 and Figure S3). Without a bias in diffusion, we found that the simulated mRNP would require an average of 110 min to reach the actin patch, which is longer than the reported lifetime of the BBC1 mRNA (t1/ 2 = 43 min, [120]) and longer than a full S. cerevisiae cell cycle. However, with even a seemingly tiny bias in the diffusion, approximated here by a single 8 nm step for the non-processive motor [121] in the direction of an actin patch once per second, we find that the simulated mRNP would require an average of 11 min to travel from the nucleus to the actin patch. This seemingly negligible bias would not be reflected by an observable change in the overall diffusion coefficient or even, necessarily, in the confinement volume. However, our modeling demonstrates that the diffusion rate of BBC1 alone is not sufficient to account for the efficient localization of this mRNA to the actin patch by simple, unbiased diffusion. Furthermore, BBC1 binding to the non- processive motor, Myo3, and the lack of any sustained directed movement are consistent with biased diffusion for efficient localization to actin patches. Indeed, in such a model the increased concentration of polarized actin filaments that arises from the actin nucleation and branching at sites of endocytosis would create a potential well where a particle exhibiting biased diffusion could be effectively trapped without being rigidly bound; sites where actin patches are clustered, such as the presumptive bud in early S phase cells, would trap mRNPs for longer periods of time. Biased diffusion by non-processive motor movement before capture in a region of the cell that is dense with actin filaments is similar to the proposed mechanism of melanosome transport in Xenopus. Single particle tracking of melanosomes transported by microtubule motors and by myosin V revealed long, microtubule- based movement interspersed with short, intermittent steps along actin filaments with frequent loss of attachment, passive diffusion, then reattachment to other actin filaments until the melanosomes were tethered in the actin-rich distal region of dendrites [122]. We believe our results favor a biased diffusion model for AP mRNA localization to actin patches. Retention of the AP mRNPs in the potential well of the actin patch would provide time for localized translation of the AP mRNAs to locally enhance actin branching and dynamics. For example, previous work in our laboratory found that in rapidly growing S. cerevisiae haploid cells, 69% of LAS17 (WASP) mRNAs are associated with polyribosomes, averaging 11 ribosomes per mRNA [123]. Assuming a translation rate of 10 amino acids per second [124], a single ,1900 base LAS17 transcript in polysomes would yield only about 10 proteins per minute. Because there are only 1–3 LAS17 transcripts per haploid yeast cell [98,99], the ability to localize LAS17 and other AP mRNAs for localized translation at sites of actin branching might confer a significant selective advantage. b-actin mRNAs and PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e31912