Nuclear speckles (NSs) are nuclear biomolecular condensates that are postulated to form by macromolecular phase separation, although the detailed underlying forces driving NS formation remain elusive. SRRM2 and SON are 2 non-redundant scaffold proteins for NSs. How each individual protein governs assembly of the NS protein network and the functional relationship between SRRM2 and SON are largely unknown. Here, we uncover immiscible multiphases of SRRM2 and SON within NSs. SRRM2 and SON are functionally independent, specifically regulating alternative splicing of subsets of mRNA targets, respectively. We further show that SRRM2 forms multicomponent liquid phases in cells to drive NS subcompartmentalization, which is reliant on homotypic interaction and heterotypic non-selective protein-RNA complex coacervation-driven phase separation. SRRM2 serine/arginine-rich (RS) domains form higher-order oligomers and can be replaced by oligomerizable synthetic modules. The serine residues within the RS domains, however, play an irreplaceable role in fine-tuning the liquidity of NSs.

Focal adhesions (FAs) are transmembrane protein assemblies mediating cell–matrix connection. Although protein liquid–liquid phase separation (LLPS) has been tied to the organization and dynamics of FAs, the underlying mechanisms remain unclear. Here, we experimentally tune the LLPS of PXN/Paxillin, an essential scaffold protein of FAs, by utilizing a light-inducible Cry2 system in different cell types. In addition to nucleating FA components, light-triggered PXN LLPS potently activates integrin signaling and subsequently accelerates cell spreading. In contrast to the homotypic interaction-driven LLPS of PXN in vitro, PXN condensates in cells are associated with the plasma membrane and modulated by actomyosin contraction and client proteins of FAs. Interestingly, non-specific weak intermolecular interactions synergize with specific molecular interactions to mediate the multicomponent condensation of PXN and are efficient in promoting FA assembly and integrin signaling. Thus, our data establish an active role of the PXN phase transition into a condensed membrane-associated compartment in promoting the assembly/maturation of FAs.

Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific rb1cc1- and atg14-conditional knockout (cKO) mice by using Ckm/Ckmm2-Cre and compared their phenotypes to those of ulk1 ulk2-conditional double-knockout (cDKO) mice. atg14-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both atg14-cKO and rb1cc1-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin⁺ SQSTM1⁺ deposits, but only those of rb1cc1-cKO mice showed TARDBP/TDP-43⁺ pathology and other features of the inclusion body myopathy–like disease we previously described in ulk1 ulk2-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP⁺ pathology. ABBREVIATIONS:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100