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.

Download full-text

Full-text

Available from: Tatjana Trcek, Aug 15, 2015
1 Follower
 · 
157 Views
  • 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.
    Neuron 10/2013; 80(3):648-57. DOI:10.1016/j.neuron.2013.10.036 · 15.98 Impact Factor
  • 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.
    Genes & development 09/2012; 26(17):1885-90. DOI:10.1101/gad.190413.112 · 12.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) controls the cytoplasmic fate of specific target mRNAs including ACTB and CD44. During neural development, IGF2BPs promote neurite protrusion and the migration of neuronal crest cells. In tumor-derived cells, IGF2BP1 enhances the formation of lamellipodia and invadopodia. Accordingly, the de novo synthesis of IGF2BP1 observed in primary malignancies was reported to correlate with increased metastasis and an overall poor prognosis. However, if and how the protein enhances metastasis remains controversial. In recent studies, we reveal that IGF2BP1 promotes the directed migration of tumor-derived cells in vitro by controlling the expression of MAPK4 and PTEN. The IGF2BP1-facilitated inhibition of MAPK4 mRNA translation interferes with MK5-directed phosphorylation of the heat shock protein 27 (HSP27). This limits G-actin sequestering by phosphorylated HSP27, enhances cell adhesion and elevates the velocity of tumor cell migration. Concomitantly, IGF2BP1 promotes the expression of PTEN by interfering with PTEN mRNA turnover. This results in a shift of cellular PtdIns(3,4,5)P 3/PtdIns(4,5)P 2 ratios and enhances RAC1-dependent cell polarization which finally promotes the directionality of tumor cell migration. These findings identify IGF2BP1 as a potent oncogenic factor that regulates the adhesion, migration and invasiveness of tumor cells by modulating intracellular signaling.
    Cell adhesion & migration 07/2012; 6(4):312-8. DOI:10.4161/cam.20628 · 3.40 Impact Factor
Show more