Fluorescence photoactivation as a tool to determine the dynamics of tau mRNA binding proteins

The axonal microtubule associated protein tau plays an important role in the development of nerve cells and in neurodegeneration. Regulation of tau expression could contribute to tau localization and function. Biochemical studies have revealed that specific mRNA-binding proteins (RBPs) interact with the tau mRNA in so-called RNA granules and may affect tau expression. Here we studied two RBPs, which are known to interact with the tau mRNA, G3BP-1 and IMP-1. Fusion constructs with photoactivatable GFP (PAGFP) were prepared and transiently expressed in neuronally differentiated PC12 cells. To determine the intracellular distribution and dynamics, focal photoactivation was performed and computer assisted tracking and morphological analyses were employed. Both G3BP-1 and IMP-1 were distributed in granular structures in PC12 cells. A fraction of the granules contained tau mRNA as determined using a molecular beacon approach. G3BP-1 containing granules were faster and exhibited a more dynamic protein exchange compared to IMP-1 containing granules. Both types of granules showed similar morphological features as judged by volumetric analysis. The data suggest that G3BP-1 and IMP-1 containing granules consist of different mRNA species and various RBPs and represent distinct types of RNA granules.