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

Here we identified the stress-induced translocation of SINTBAD to SINT-speckles, as revealed by immunofluorescence and cell fractionation experiments. These SINT-speckles form a subcellular compartment with no significant overlap to other characterized MLOs (Darling et al., 2018) . SINTBAD contains two lowcomplexity domains containing 12.9% Gln (residues 106-346) and 27.7% Pro (residues 340-535). Core components of constitutive SINT-speckles such as ABIN2 and AZI2, and also the inducible interactor SINTBAD, are predicted to harbor long intrinsically disordered regions ( Figure 9A , Table S1 ), which are typical for MLO-resident proteins (Elbaum-Garfinkle et al., Nott et al., 2015; Uversky and Dunker, 2010) . Accordingly, about one-third of the SINT-speckle proteins (47 from 150) are predicted to have >40% disordered regions (Table S1 ). The mechanisms controlling the formation of constitutive SINT-speckles are not known and might involve post-translational modifications such as ULK1/2-mediated phosphorylation. Another possible mechanism could involve the bridging of two ABIN2 dimers by binding to M1-linked tri-ubiquitin chains, which might facilitate ABIN2 assembly to higher-order signaling complexes (Lin et al., 2017) . Also, changes in the relative expression levels of ABIN2 speckle components affect the formation of these MLOs, explaining the abundant finding that expression of a given protein such as ABIN2 or AZI2 can affect the intracellular localization of its interactors. This behavior is characteristic for MLO formation, and accordingly, also overexpression of SG components such as TIA1 or G3BP1 is sufficient to trigger formation of SGs (Kedersha and Anderson, 2007) . This implies that physiological variations in the amounts of SINTspeckle proteins can already affect speckle formation. Regulation of ABIN2 protein levels occurs in the presence of increased glucose levels or by the kinases TPL2 or IKKa/b (Chen et al., 2013; Leotoing et al., 2011; Nanda et al., 2018) , and it will thus be interesting to investigate whether these situations will affect the formation of SINT-speckles. The formation of inducible SINT-speckles is regulated by several mechanisms, as schematically shown in Figure 9B . SINT-speckle formation is triggered by the acetyltransferase KAT2A. A recent study showed the relevance of acetylation of low-complexity domains for the formation of SGs (Saito et al., 2019) , and it will be interesting to investigate whether the enzymatic activity of KAT2A contributes to its ability to promote inducible SINT-speckle formation. Formation of inducible SINT-speckles is antagonized by the kinase activity of its component ULK1, raising the possibility of an autoregulatory control of speckle homeostasis. Interestingly, a recent study showed that, vice versa, SINTBAD Figure 6 . Continued cotransfected to express FLAG-KAT2A and hemagglutinin (HA)-SINTBAD were treated the same way. Scale bar, 10 mm. The right part schematically summarizes the intracellular localization of the proteins.