Article

An Unbiased Analysis Method to Quantify mRNA Localization Reveals Its Correlation with Cell Motility

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Cell Reports (Impact Factor: 8.36). 02/2012; 1(2):179-84. DOI: 10.1016/j.celrep.2011.12.009
Source: PubMed

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.

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    • "This control experiment demonstrates that FISH-STICs probes under the conditions we report are mRNA-specific and do not have intrinsic background binding. We next analyzed FISH-STIC images to quantify Actb and Actg mRNA distribution within the same cell by measuring the polarization index and dispersion index as reported by Park et al. (2012). These indexes quantify the distribution of an mRNA within the cell in relationship to the cell's morphology. "
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    • "This also raises the intriguing question of whether translation from monosomes, rather than polysomes, may be more common in distal neuronal compartments where there could be demand for a few highly localized proteins. New high-resolution single molecule detection methods (Cajigas et al., 2012; Park et al., 2012) and live-imaging methods for translation (Chao et al., 2012) will be valuable when answering these sorts of questions. 5. What mRNAs Are Translated in Subcellular Compartments In Vivo? With the advent of TRAP (translating affinity purification) technology (Heiman et al., 2008) it will be possible in the future to answer this question in specific neuronal compartments of specific subsets of neurons. "
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    • "(A) Live-cell TIRF images of wild-type and ZBP1 knockout fibroblasts with TagRFPt-labeled b-actin mRNA and free GFP as the cytoplasmic marker. Corresponding polarization indices are shown, based on a reported algorithm that assesses asymmetry by computing the intensity-weighted centroids of mRNA and cytoplasmic GFP (Park et al. 2012). (B) The average polarization index of b-actin mRNA distribution was significantly lower in cells without ZBP1. "
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