Linear ubiquitination induces NEMO phase separation to activate NF-KB signaling

The NF-KB essential modulator NEMO is the core regulatory com-ponent of the inhibitor of KB kinase complex, which is a critical checkpoint in canonical NF-KB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-113, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-113. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional rele-vance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is de-fective in mediating IL-113-induced NF-KB activation.