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

Many proteins participating in the induction of innate immune response signaling have a propensity to form inducible polymers or filaments, which show a reduced solubility in standard lysis buffers (David et al., 2018; Pellegrini et al., 2018; Vajjhala et al., 2017) . To test the possible impact of cellular stress on the solubility of SINTBAD, HeLa cells were exposed to heat shock for different periods, followed by fractionation into a cytosolic and a nuclear/insoluble fraction representing not only chromatin proteins but also poorly soluble or aggregated proteins. A western blot analysis revealed that treatment with heat shock triggered the time-dependent transition of SINTBAD from the soluble to the nuclear/insoluble fraction, whereas this dynamic redistribution between the fractions did not occur for the SINTBAD-related proteins AZI2 or TANK ( Figure 1A) . A similar inducible redistribution of SINTBAD also occurred in arsenite-treated 293T cells ( Figure 1B) , showing that various stressors can trigger the relocation of SINTBAD in different cell types. It was also interesting to test whether SINTBAD translocation to the insoluble fraction is also seen in response to further different stimuli representing inflammatory conditions or osmotic or proteotoxic stress. These results show that most adverse agents, with the exception of inflammatory stimuli, triggered translocation of SINTBAD to the nuclear/insoluble fraction ( Figure 1C ). This behavior was seen for the endogenous proteins and also for the adapter proteins when expressed at moderate levels (Figures S1A and S1B).