ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation

Saul, Vera Vivian; Seibert, Markus; Krüger, Marcus; Jeratsch, Sylvia; Kracht, Michael; Schmitz, Michael Lienhard

(D) Double-deficient (sgAZI2/sgSINTBAD) U2OS cells stably expressing FLAG-SINTBAD or transfected to express moderate amounts of FLAG-AZI2 or FLAG-TANK were treated with arsenite (0.5 mM, 1 h) or heat shock (43 C, 1 h) and analyzed by indirect immunofluorescence using anti-FLAG antibodies. Nuclear DNA was stained with Hoechst; scale bars, 10 mm. The percentage of cells showing a localization as displayed in the figure is indicated. Figures S3A and S3B ). These cells were used to investigate its distribution in cells exposed to arsenite, heat shock, sorbitol, or Earle's balanced salt solution (EBSS) starvation medium. Untreated control cells showed SINTBAD as a largely cytosolic protein, whereas the various cell stressors triggered the formation of speckles occurring mainly in the cytosol and to a minor extent in the nucleus ( Figures 1D and S4 ), reflecting the transition of SINTBAD from the cytosolic to the nuclear/insoluble fraction. Staining of FLAG-tagged TANK did not reveal any arsenite or heat shockinduced changes of its cytosolic localization, whereas AZI2 already showed a speckled distribution in unstressed cells that was not further influenced by the indicated stressors ( Figure 1D ).