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

The authors declare no competing interests. Elbaum-Garfinkle, S., Kim, Y., Szczepaniak, K., Chen, C.C., Eckmann, C.R., Myong, S., and Brangwynne, C.P. (2015) . The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl. Acad. Sci. U S A 112, 7189-7194. Fig. S5 . Disaggregation of heat shock-induced SINT-speckles is independent of de novo protein expression and autophagy, Related to Figure 3 . (A) SINTBAD-deficient U2OS cells reconstituted with Flag-SINTBAD were left untreated or exposed to heat shock. Indicated cells were allowed to recover from heat shock at 37 °C for 3 h in the absence or presence of the translation inhibitor anisomycin (5 µg/ml). (B) SINTBAD-deficient U2OS cells stably expressing Flag-SINTBAD were left untreated or exposed to heat shock, followed by a 3 h long recovery period at 37 °C. Treatments and recovery were performed in the presence of the indicated autophagy inhibitors (0.5 µM Bafilomycin A, 20 mM NH 4 Cl, 20 µM Chloroquine). The percentage of cells showing the displayed phenotype is indicated. et al., 2013)) into the pEGFP-C2 vector (Clontech); PCR-amplified ABIN2 from pCAGGS-E-hABIN2, that was kindly provided by Dr. R. Beyaert (VIB-Ghent University, Belgium, (Van et al., 2001) ) was subcloned with a N terminal HA-tag into pcDNA6 (Invitrogen); pcDNA3.1-HA-ULK1 was obtained from Dr. S. H. Tooze (The Francis Crick Institute, London, UK, (Joachim et al., 2015) ) and its kinase-inactive K46I mutant was generated by site-directed mutagenesis (QuikChange II XL, Agilent, (Chan et al., 2009) ). Flag-KAT2A was from Dr.