Super-Resolution Fluorescence Imaging Reveals Nanoscale Organization of Stress Granule

Abstract

In response to environmental stresses, cytoplasmic mRNAs assemble and form stress granules (SGs) accompanied by several proteins affecting mRNA functions. SGs have been proposed to play an important role in mRNAs remodeling for repression of translation. However, the underlying mechanism remains unclear, since conventional fluorescence imaging cannot resolve the detailed distributions of SGs components due to the diffraction limit. Here, we investigated nanoscale organizaion of SGs by stochastic optical reconstruction microscopy (STORM), which provided us super-resolution images with spatial resolution of ∼20 nanometers in the lateral direction and of ∼60 nanometers in the axial direction.Super-resolution imaging revealed that mRNAs in SGs were highly localized to tiny compartments with a diameter of less than 100 nanometers, whereas they were elusive in conventional fluorescence imaging. In mature SGs, the number of these compartments was higher than in small SGs, but the size of these compartments showed little difference. The result demonstrated that the growing process of SGs resulted from the assembly of tiny compartments. Furthermore, multicolor super-resolution imaging showed that some SG associated proteins colocalized with mRNAs and others did not. Since SGs components were densely packed within several micrometers radius, we could not observe the differences by conventional fluorescence imaging. These results suggested that SGs have highly organized composition that would be responsible for the physiological functions in cellular stress responses and that super-resolution imaging technique is a powerful tool to investigate the detailed organization of densely packed granular architectures in cells.