Tanja Mittag, Ph.D., Associate Member, Department of Structural Biology, Faculty of the Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital

Phase separation and mesoscale assembly for functional compartmentalization

Liquid-liquid phase separation of proteins leads to demixing from solution and results in a dense, protein-rich phase, which co-exists with a light phase depleted of protein. Recent findings support a model in which phase separation is the biophysical driving force for the formation of membrane-less organelles in cells, such as stress granules, nucleoli and nuclear speckles. Current open questions are: (i) How is phase separation propensity encoded in the protein sequence, (ii) are dense liquid droplets used as reaction compartments in the cell, and (iii) is physiological phase separation disrupted in disease states? To address these, we study two systems, the tumor suppressor Speckle-type POZ protein (SPOP) and the RNA-binding protein hnRNPA1.

SPOP, a substrate adaptor of a ubiquitin ligase, localizes to different liquid membrane-less organelles in the cell nucleus, where it encounters its substrates, but it is never found diffuse in the cell. However, its recruitment mechanism to these organelles is not understood. Here, we show that SPOP undergoes LLPS with substrate proteins, and that this mechanism underlies its recruitment to membrane-less organelles. Multivalency of SPOP and substrate for each other drive their ability to phase separate. We present evidence that the SPOP/substrate assemblies are active ubiquitination compartments in vitro and in cells. SPOP cancer mutations reduce the propensity for phase separation. We propose that SPOP has evolved a propensity for phase separation in order to target substrates localized in membrane-less compartments.

Recent mutagenesis experiments have revealed the importance of aromatic residues for the ability of low-complexity regions (LCRs) of RNA-binding proteins to undergo LLPS. Here, we investigate the interactions that mediate phase separation of the intrinsically disordered LCR of hnRNPA1. Phase separation of hnRNPA1 promotes the fibrillization of mutants of hnRNPA1 that cause ALS and other neurodegenerative diseases. We find that aromatic side-chains cluster and lead to compaction of the LCR, and that this compaction is coupled to LLPS. Understanding the interactions that mediate phase separation has the potential to provide mechanistic insight into membrane-less compartmentalization in cells. 

Host: Assoociate Professor Frans Mulder, iNANO & Dept. of Chemistry, Aarhus University