Fig 2.

Phosphorylation-independent inhibition of stress granule assembly by MK-STYX. (A, C) Representative examples of the subcellular distribution patterns of GFP-tagged G3BP-149 mutants. HeLa cells cotransfected with expression vectors for S149A-G3BP and wild-type MK-STYX showed fewer cells with stress granules. The overexpression of S149A-G3BP alone resulted in stress granule assembly. Cells cotransfected with S149A-G3BP and the active MK-STYX mutant accumulated larger perinuclear granules. Cells expressing S149E-G3BP mutant alone or in the presence of MK-STYX did not accumulate stress granules, whereas cells expressing S149E-G3BP and the MK-STYX active mutant accumulated smaller granules that were more dispersed throughout the cytoplasm. Merged images show the location of GFP-tagged G3BP-1 mutants (green) relative to DAPI-stained nuclei (blue). Scale bar = 10 μm. (B, D) HeLa cells were cotransfected with expression vectors for GFP-tagged G3BP-149 mutants, and either MK-STYX or MK-STYX active mutant. Cells were analyzed 24 h post-transfection for G3BP-induced stress granule assembly by fluorescence microscopy, after staining DAPI to reveal the nuclei. Cells were scored for the presence or absence of stress granules. Three replicate experiments were performed (n = 100); error bars indicate the SEM.