Quantifying Actin mRNA Expression with Super Resolution Fluorescence Microscopy

Abstract

The copy number of a specific mRNA and its subcellular localization in cells is a mode by which cells can regulate protein synthesis. Beta-actin mRNA is transported to filopodia upon serum stimulation, regions of cell growth and movement where actin polymerization is essential (1). The copy number of mRNA molecules directly translates to the amount of actin monomers that are synthesized. Quantifying the actin mRNA copy numbers provides a measure of how polymerization of actin is regulated.To this extent, we apply stochastic super-resolution microscopy with photoswitchable fluorescent probes combined with single-molecule localization (2,3) for quantitative studies of mRNA concentration and localization in eukaryotic cells. For this purpose, we apply various labelling approaches for mRNA, including well-established methods such as fluorescence in-situ hybridization (FISH) (4). Focussing on actin mRNA, we derive quantitative information on copy numbers, and we apply coordinate-based algorithms to analyze our single-molecule super-resolution data.References(1) Ben-Ari et al. (2010) J Cell Sci, 123, 1761-1774.(2) Betzig et al. (2006) Science, 313, 1642-1645.(3) Heilemann et al. (2008) Angew. Chemie, 47, 6172-6176.(4) Levskey, J. M.; Singer, R. (2003) Journal of Cell Science, 116, 2833-2838.