Actin Patch mRNA Localization and Dynamics Figure 4. 2D dynamics of actin patch mRNA localization. (A) Montage of a time-lapse video of LAS17:MS2 (green) localization in a haploid, early S phase cell with actin patch accumulation at the presumptive bud indicated by FM4–64 staining (red). Scale bar is 2 mm. (B) The trajectory of the mRNA particle in panel (A) is shown on a grayscale image of the first timepoint (0 s) with black-white inversion. Time is encoded by color in the heatmap. (C) Montage of a time-lapse video of LAS17:MS2. Scale bar is 5 mm. Brightness of the latter timepoints in the montage was adjusted relative to the earliest timepoints for display purposes in order to compensate for photobleaching. (D) The trajectory of the mRNA particle from panel (C) is displayed as in panel (B). (E) Change in the diffusion coefficient of the mRNA particle in panels (C) and (D) over time. The diffusion coefficient for short time lags (t = 4) is graphed with time encoded by color as in panel (D). D was extracted from 4 non-overlapping segments of the trajectory of length 8.992 s, or 16 timepoints. The highest estimated D value, 0.00138 mm2/s, leads to an approximate standard deviation of 5.6361023 mm2/s [135,136]. doi:10.1371/journal.pone.0031912.g004 practice, the DH-PSF system and image processing allowed us to determine the location of single resolved, diffraction-limited BBC1 mRNPs to an average spatial precision of 25 nm in x and y and 50 nm in z with 50 ms exposure times, allowing for high confidence studies of single mRNP dynamics. We chose to focus our further investigation of the dynamics of Myo3-associated AP mRNAs on BBC1 because of its distinct pattern of localization to the actin patch (Figures 3A and 3B) and good signal to noise for the BBC1:MS2 mRNP relative to the background MS2-CP-3xEGFP signal in the cytoplasm. A representative, single-particle trajectory of BBC1:MS2 lasting approximately 35 seconds is displayed in Figure 5B. The mRNP exhibits considerable movement within a relatively confined area, moving a total distance of 28.9 mm within a radius of ,300 nm from 0–29 seconds, after which the particle exhibits diffusive behavior, moving a total distance of 10.8 mm in just 5 seconds. As discussed below, these prolonged confinements are likely due to an association with actin patches; these events are distinct from the transient (,1 s) confinements observed in this system and in our previous studies of ARG3 [107]. A statistical test of the probability that this particle was confined (Figure 5C; [107]) provided evidence for confinement for the majority of the observation period with much of the trajectory surpassing the 95% confidence threshold (Figure 5C, gray line), that is, the probability that the motion is Brownian, Y, is very small. The portion of the trajectory from 29.25 s to 34.45 s appeared to be consistent with simple diffusion; analysis of this period by calculating the mean squared displacement (MSD) of the particle over time for short time lags (Figure 5D) resulted in a linear fit of the MSD plot (R2 = 0.996), consistent with the BBC1 mRNP being diffusive during this period with an average D = 0.038 mm2/s. PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31912