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Figure 8. Nascent β-actin Microdomains Form in Close Proximity to Docked RNA Granules In Vivo(A) Dual-channel simultaneous time-lapse images of Cy5-RNA and Venus-β-actin FRAP. The axon morphology was estimated by capturing a mRFP image before photobleaching and after time-lapse acquisition, which are overlaid on the Cy5-RNA images (gray scale, top) at time points 0″ and 300″, respectively. Axon outline of the mRFP image captured after time-lapse acquisition was used as an approximation for time points 10″ to 300″. Venus-β-actin signal recovery after photobleaching is illustrated by the fluorescence heatmaps (middle). The bottom row presents the overlays of Cy5-RNA (cyan) and Venus-β-actin FRAP (magenta).(B) Enlarged images of area signified by the arrowhead in (A). The images of Cy5-RNA (cyan) and Venus-β-actin FRAP (magenta) are individually presented on the left and in the middle columns. Images on the right display image overlays. The FRAP signal positions at 30 s closely resemble the localization of RNA at 10 s.(C) Cy5-RNA time series were compiled into z stacks and computed for the median signal intensities as a representation of RNA dwell time. The resulting images were then used to compute Pearson’s correlation coefficient (R) with the Venus-β-actin cumulative recovery signal. The averages of R(observed) were significantly higher than averages of R(random) yielded from 1,000 random images scrambled from each original axon image (t8 = 11.55, p < 0.0001, paired t test).Scale bars, 5 μm for (A) and 1 μm for (B). Image published in: Wong HH et al. (2017) © 2017 The Authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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