Genetically encoded reporters of actin filament organization in living cells and tissues

Abstract

SUMMARYThe cytoskeletal protein actin is crucial for cell shape and integrity throughout eukaryotes. Actin filaments perform essential biological functions, including muscle contraction, cell division and tissue morphogenesis. These diverse activities are achieved through the ability of actin filaments to be arranged into diverse architectures, but a detailed appreciation of the dynamic organizational state of the actin filaments has been hindered by available tools. Fluorescence polarization microscopy is uniquely placed for measuring actin organization by exploiting the sensitivity of polarized light excitation to the orientation of fluorophores attached to actin filaments. By engineering constrained fluorescent protein fusions to widely used actin localization reporters, we have succeeded in developing novel genetically-encoded reporters for non-invasive, quantitative measurements of actin filament organization in living cells by fluorescence polarization microscopy. We show examples of actin organization measurements in living mammalian cells in culture, as well asin vivoin fission yeast,C. elegansandDrosophila.