Actin Patch mRNA Localization and Dynamics Figure 5. 3D super-localization of mRNA particle dynamics using a DH-PSF setup. (A) Schematic of the DH-PSF setup using a spatial light modulator (SLM) exhibiting a phase mask [110] that produces the double-helix point spread function in the imaging plane (EMCCD camera). A single, diffraction-limited point of light shown in the standard imaging plane is detected as two spots in the DH-PSF imaging plane with z-position encoded in the angle between the two lobes. (B) 3D super-localization trajectory of a single BBC1:MS2 particle at 50 ms temporal resolution. Time is encoded by color as displayed in the heatmap. (C) Plot of log Y, a measure of the probability that a particle is confined during a portion of the trajectory, for the particle trajectory from panel (B) with time encoded by color. Portions of the trajectory falling below the 95% confidence threshold (dark grey line) are confined. (D) Mean-squared displacement (MSD) plot of the portion of the trajectory in B and C from 29.25 s to 34.45 s, plotted versus time for short time lags. doi:10.1371/journal.pone.0031912.g005 Deletion of MYO3 Decreases the Proportion of Stationary BBC1 mRNP Trajectories Although actin patches are dynamic objects [92,94,97], the temporal resolution of our measurements (50 ms) make it possible that short term capture in a Myo3- or actin –dependent manner may be detected. Because disruption of MYO3 and addition of latrunculin B decreased the proportion of AP mRNAs localized to actin patches (Figure 3), we wanted to evaluate the influence of Myo3 and F-actin on the ability of mRNPs to be captured and held ‘‘stationary’’ in cells using statistical tests that we have derived previously for the identification of stationary objects ([107]; Table 1). A trajectory was defined as being stationary if the root mean squared (RMS) displacement at the fourth time lag (0.2 s) was less than the total localization precision of the measurement Table 1. Differential Proportion of ARG3 and BBC1 mRNP Dynamic States. Type of Motion Directed Stationary and Confined Diffusive ARG3* BBC1 BBC1+latB BBC1 myo3D 4% 21% 75% 2.2% 34.4% 63.4% 2.0% 27.3% 70.7% 3.6% 18.2% 78.2% *ARG3 data was published in Thompson et al. [107]. doi:10.1371/journal.pone.0031912.t001 PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31912