An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility - PubMed (original) (raw)

An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility

Hye Yoon Park et al. Cell Rep. 2012.

Abstract

Localization of mRNA is a critical mechanism used by a large fraction of transcripts to restrict its translation to specific cellular regions. Although current high-resolution imaging techniques provide ample information, the analysis methods for localization have either been qualitative or employed quantification in nonrandomly selected regions of interest. Here, we describe an analytical method for objective quantification of mRNA localization using a combination of two characteristics of its molecular distribution, polarization and dispersion. The validity of the method is demonstrated using single-molecule FISH images of budding yeast and fibroblasts. Live-cell analysis of endogenous β-actin mRNA in mouse fibroblasts reveals that mRNA polarization has a half-life of ~16 min and is cross-correlated with directed cell migration. This novel approach provides insights into the dynamic regulation of mRNA localization and its physiological roles.

Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Quantification of ASH1 mRNA Distributions in Wild-Type and ΔSHE2 Budding Yeasts

(A) A schematic showing the polarization vector of mRNA distribution pointing from the centroid of the cell to the centroid of the single mRNA positions. (B) Overlay image of ASH1 mRNA molecules (magenta), and nuclei (blue) in a wild-type cell. ASH1 mRNAs localize to the daughter bud tip. Scale bar represents 1 μm. (C) Scatter plot of polarization index in x axis and dispersion index in y axis for wild-type cells. (D) Overlay image of ASH1 mRNA molecules, and nuclei in a ΔSHE2 cell. Deletion of She2p causes a complete delocalization of ASH1 transcripts. Scale bar represents 1 μm. (E) Scatter plot of polarization index versus dispersion index for ΔSHE2 cells. The red arrow heads indicate the data points for the cells shown in (B) and (D). See also Figure S1.

Figure 2

Figure 2. Comparison of GAPDH and β-Actin mRNA Distributions in Chicken Embryonic Fibroblasts

(A) FISH image of GAPDH mRNA (red), and β-actin mRNA (green). Scale bar represents 10 μm. (B) Scatter plot of polarization index for GAPDH mRNA in x axis and β-actin mRNA in y axis. (C) Scatter plot of dispersion index for GAPDH mRNA in x axis and β-actin mRNA in y axis. Red dashed lines indicate cells that have the same index for GAPDH and β-actin mRNA. See also Figure S2.

Figure 3

Figure 3. Localization of β-Actin mRNA in a Migrating Cell

(A) Time-lapse images of a mouse embryonic fibroblast. The color map shows the fluorescence intensity of MCP-GFP labeling endogenous β-actin transcripts divided by the intensity of red fluorescent cytoplasmic dye. Yellow arrows show the polarization vector of mRNA distribution, and red arrows show the protrusion vector of the cell. (B) Mean auto-correlation curves of the polarization vector (black) and the protrusion vector (red) (n = 11 cells). The shaded areas indicate 95% confidence intervals. (C) Mean cross-correlation curve of the polarization vector and the protrusion vector (black curve) and 95% confidence interval (gray area) for n = 11 cells. (D) Migration distance as a function of the time-average of the polarization index over 2 hr. The correlation coefficient between the polarization index and the migration distance is 0.75. See also Figure S3 and Movie S1.

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