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


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.

Download full-text


Available from: Tatjana Trcek
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to its use. We devised a means to use commercially synthesized oligonucleotides as RNA-FISH probes without further modification and show that such probes work well for detection of RNA in cultured cells. This approach can bind a high concentration of fluorescent ODN to a short stretch of an RNA using commercial DNA synthesis outlets available to any laboratory. We call this approach for creating in situ hybridization probes Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes (FISH-STICs). We demonstrate that one FISH-STIC probe can detect mRNA molecules in culture, and that probe detection can be improved by the addition of multiple probes that can be easily adapted for robust mRNA quantification. Using FISH-STICs, we demonstrate a nonoverlapping distribution for β-actin and γ-actin mRNA in cultured fibroblasts, and the detection of neuron-specific transcripts within cultured primary hippocampal neurons.
    Preview · Article · Dec 2013 · RNA
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The elaborate morphology of neurons together with the information processing that occurs in remote dendritic and axonal compartments makes the use of decentralized cell biological machines necessary. Recent years have witnessed a revolution in our understanding of signaling in neuronal compartments and the manifold functions of a variety of RNA molecules that regulate protein translation and other cellular functions. Here we discuss the view that mRNA localization and RNA-regulated and localized translation underlie many fundamental neuronal processes and highlight key issues for future experiments.
    Full-text · Article · Oct 2013 · Neuron
  • Source
    • "(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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell's cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports β-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of β-actin mRNA and protein localization by tethering the mRNA to a specific location-in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes.
    Full-text · Article · Sep 2012 · Genes & development
Show more