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

Spatial and temporal organization of cellular signaling pathways heavily relies on compartmentalization. Structural surfaces for signal transduction can be formed by membranes, components of the cytoskeleton, membrane-surrounded organelles, or membrane-less organelles (MLOs). These MLOs occur in the cytoplasm and nucleoplasm of eukaryotic cells and have recently also been discovered in bacteria (Uversky, 2017; Brangwynne, 2013; Al-Husini et al., 2018) . These liquid-like subcellular compartments are believed to be formed by phase separation (Boeynaems et al., 2018) , a process that involves the spontaneous separation of a supersaturated solution into a dense and a dilute phase. The liquid-like nature of MLOs allows fusion and fission events and a dynamic exchange of components. MLOs comprise a variety of subcompartments including nucleoli, promyelocytic leukemia (PML) nuclear bodies, P bodies, and stress granules (SGs) (Boeynaems et al., 2018; Darling et al., 2018; Wheeler and Hyman, 2018) . The dense phase inside MLOs contains a high concentration of proteins, which may facilitate biochemical reactions and control signaling thresholds from these very crowded environments (Boeynaems et al., 2018; Gomes and Shorter, 2018; Woodruff et al., 2017) . In addition, MLOs can act as dynamic buffers for RNAs and proteins. This buffering function also serves as a passive noise filter and thus reduces the inherent randomness of chemical reactions (Saunders et al., 2012) . Different MLOs have a variety of functions ranging from the expression of rRNAs and pre-assembly of ribosomes (nucleoli) to the organization of the spindle apparatus (centrosomes) and mRNA splicing (splicing speckles) (Bernardi and Pandolfi, 2007; Boulon et al., 2010; Brangwynne, 2013; Hyman et al., 2014) . Aberrant forms of MLOs occur upon failure of the protein quality control system or by mutation of MLO-resident proteins, often causing age-related diseases such as amyotrophic lateral sclerosis, inclusion body myopathy, and frontotemporal dementia (Hock and Polymenidou, 2016; Malinovska et al., 2013; Ramaswami et al., 2013; Taylor et al., 2016) . Furthermore, various viruses hijack MLO proteins to aid in their replication (Dhillon and Rao, 2018; Mö ller and Schmitz, 2003; Nakagawa et al., 2018; Reineke and Lloyd, 2013) . MLOs are dynamically formed by an interplay between RNA and intrinsically disordered proteins (IDPs) that typically harbor low-sequence-complexity domains enriched in polar side chains (Arg, Gln, Glu, Ser, Lys) or structure-breaking amino acids (Gly, Pro) (Uversky and Dunker, 2010) . The dynamic formation of MLOs can be regulated by post-translational modifications such as acetylation, SUMOylation, or phosphorylation (Bernardi and Pandolfi, 2007; de la Vega et al., 2011; Saito et al., 2019; Wippich et al., 2013) or by environmental cues such as changes in temperature, pH, or osmolarity (Uversky, 2017) . Interestingly, a small interfering RNA (siRNA) screen identified several kinases as regulators of MLO formation, including the Ser/Thr kinase TBK1 as a regulator of splicing speckles (Berchtold et al., 2018) .