| Popis: |
Stress granules (SGs) are cytosolic, nonmembranous RNA-protein (RNP) complexes that form in the cytosol of many cells under various stress conditions and can integrate responses to various stressors. Although physiological SG formation appears to be an adaptive and survival-promoting mechanism, inappropriate formation or chronic persistence of SGs has been linked to aging and various neurodegenerative diseases. The quantitative monitoring of the dynamics of SG components in living nerve cells can therefore be an important tool for identifying conditions that disrupt SG function and lead to disease-related attacks in the cells. Here, we describe a method for the quantitative determination of the distribution and shuttling dynamics of components of SGs in living model neurons by fluorescence decay after photoactivation (FDAP) measurements using a standard confocal laser scanning microscope. The method includes lipofection of photoactivatable green fluorescent protein (paGFP) fused to an SG protein of interest in a neural cell line, differentiation of the cells into a neuronal phenotype, focal activation using a blue diode (405 nm), and recording of decay curves with a 488 nm laser line. By modeling the decay measurements with FDAP functions, the approach enables estimating the residence time of the SG protein of interest, determining the proportion of the respective component in SGs, and the detection of possible changes after experimental manipulation. |
