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

Further costaining experiments revealed that some interactors can trigger the formation of SINT-speckles even in unstressed cells. An example of such a positive regulator is the lysine acetyltransferase KAT2A. Expression of this predominantly nuclear protein triggered the formation of SINT-speckles even in unstressed cells and also increased the fraction of nuclear speckles ( Figure 6A ). Another interactor triggering the formation of SINT-speckles was ABIN2, as its expression resulted in SINT-speckle formation even in unstressed cells ( Figure 6B ). The dominant effect of ABIN2 also occurred for PTPN23 ( Figure 6B , lower) and resembles that of AZI2, which was sufficient to trigger incorporation of SINTBAD into speckles (see Fig Figure S10 ). A kinase-inactive ULK1 K46I point mutant (Chan et al., 2009) , in contrast to the ULK1 wild-type, formed speckles upon cellular stress when expressed alone ( Figure 6D ). Coexpression of the kinase-inactive mutant with SINTBAD resulted in the formation of SINTspeckles even in unstressed cells, emphasizing the importance of ULK1 kinase activity for controlling SINTspeckle formation. To determine the consequences of ULK knockdown on SINT-speckle formation we interfered with the expression of ULK1 and also ULK2, as both kinases can have redundant functions (Lee and Tournier, 2011; Li et al., 2016) . Loss of both kinases already triggered the formation of SINTspeckles even in unstressed cells ( Figure 6E ), supporting the finding that constitutive ULK signaling is important for restriction of SINT-speckle formation. These kinase-dependent regulatory processes could enable regulation of SINT-speckle formation, as MLOs are highly dynamic and their formation is typically also regulated during the cell cycle (Rai et al., 2018) . This also applies to SINT-speckles, which do not occur in unstressed or stressed mitotic cells ( Figure 6F ).