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

To test the reversibility of inducible speckle formation, cells were exposed to heat shock and then further grown at 37 C to follow the fate of speckles over time. The size and number of speckles decreased in a timedependent manner, and speckles were not detectable after 3 h of cell recovery, as revealed by fluorescence microscopy ( Figure 3A ) and its quantitative analysis ( Figure 3B ). The resolution of speckles occurred also in the absence of de novo protein synthesis ( Figure S5A ) and in the presence of different lysosome inhibitors ( Figure S5B ), suggesting that lysosomal degradation and autophagic processes do not significantly contribute to this process. As chaperone function prevents aberrant phase transition of SGs (Mateju et al., 2017) it was interesting to test whether well-characterized inhibitors of HSP70 (VER155008, Pifithrin-m) or HSP90 (geldanamycin, radicicol) affect SINTBAD localization (Massey et al., 2010; Roe et al., 1999) . Inhibition of HSP70 or HSP90 function resulted in the formation of speckles even in the absence of stress signals ( Figure 3C ), revealing the importance of continuous chaperone function for the cytosolic localization of SINTBAD. Coimmunoprecipitation experiments showed the interaction between SINTBAD and HSP70, which was impaired under thermal stress conditions ( Figure 3D ). The interaction between SINT-BAD and HSP90 was only detectable after coexpression of TBK1 ( Figure 3E ), suggesting a rather indirect interaction that can be controlled by the relative abundance of the known HSP90 interactor TBK1 (Yang et al., 2006) or that the interaction is phosphorylation dependent.