In recent years, it has become clear that intrinsically disordered regions (IDRs) of RBPs, and the structure of RNAs, often contribute to the condensation of RNPs. To understand the transcriptomic features of such RNP condensates, we’ve used an improved individual nucleotide resolution CLIP protocol (iiCLIP), which produces highly sensitive and specific data, and thus enables quantitative comparisons of interactions across conditions (Lee et al., 2021). This showed how the IDR-dependent condensation properties of TDP-43 specify its RNA binding and regulatory repertoire (Hallegger et al., 2021). Moreover, we developed software for discovery and visualisation of RNA binding motifs that uncovered common binding patterns of RBPs on long multivalent RNA regions that are composed of dispersed motif clusters (Kuret et al, 2021). Finally, we used hybrid iCLIP (hiCLIP) to characterise the RNA structures mediating the assembly of Staufen RNPs across mammalian brain development, which demonstrated the roles of long-range RNA duplexes in the compaction of long 3’UTRs. I will present how the combined analysis of the characteristics of IDRs in RBPs, multivalent RNA regions and RNA structures is required to understand the formation and functions of RNP condensates, and how they change in diseases.

Interfaces and membranes are ubiquitous in cellular systems across various scales. From lipid membranes to the interfaces of biomolecular condensates inside the cell, these borders not only protect and segregate the inner components from the outside world, but also are actively participating in mechanical regulation and biochemical reaction of the cell. Being part of a living system, these interfaces (membranes) are usually active and away from equilibrium. Yet, it's still not clear how activity can tweak their equilibrium dynamics. Here, I will introduce a model system to tackle this problem. We put together a passive fluid and an active nematics, and study the behavior of this liquid-liquid interface. Whereas thermal fluctuation of such an interface is too weak to be observed, active stress can easily force the interface to fluctuate, overhang, and even break up. In the presence of a wall, the active phase exhibits superfluid-like behavior: it can climb up walls -- a phenomenon we call activity-induced wetting. I will show how to formulate theories to capture these phenomena, highlighting the nontrivial effects of active stress. Our work not only demonstrates that activity can introduce interesting features to an interface, but also sheds light on controlling interfacial properties using activity.

Living cells contain millions of enzymes and proteins, which carry out multiple reactions simultaneously. To optimize these processes, cells compartmentalize reactions in membraneless liquid condensates. Certain features of cellular condensates can be explained by principles of liquid-liquid phase separation studied in material science. However, biological condensates exist in the inherently out of equilibrium environment of a living cell, being driven by force-generating microscopic processes. These cellular conditions are fundamentally different than the equilibrium conditions of liquid-liquid phase separation studied in materials science and physics. How condensates function in the active riotous environment of a cell is essential for understanding of cellular functions, as well as to the onset of neurodegenerative diseases. Currently, we lack model systems that enable rigorous studies of these processes. Living cells are too complex for quantitative analysis, while reconstituted equilibrium condensates fail to capture the non-equilibrium environment of biological cells. To bridge this gap, we reconstituted a DNA based membraneless condensates in an active environment that mimics the conditions of a living cell. We combine condensates with a reconstituted network of cytoskeletal filaments and molecular motors, and study how the mechanical interactions change the phase behavior and dynamics of membraneless structures. Studying these composite materials elucidates the fundamental physics rules that govern the behavior of liquid-liquid phase separation away from equilibrium while providing insight into the mechanism of condensate phase separation in cellular environments.

Biologists have recently come to appreciate that eukaryotic cells are home to a multiplicity of non-membrane bound compartments, many of which form and dissolve as needed for the cell to function. These dynamical “condensates” enable many central cellular functions – from ribosome assembly, to RNA regulation and storage, to signaling and metabolism. While it is clear that these compartments represent a type of separated phase, what controls their formation, how specific biological components are included or excluded, and how these structures influence physiological and biochemical processes remain largely mysterious. I will discuss recent experiments on phase separated condensates both in vitro and in vivo, and will present theoretical results that highlight a novel “magic number” effect relevant to the formation and control of two-component phase separated condensates.

Biomolecular condensation is a mechanism for controlling cell organization. Many condensates are rich in nuclei acids such as RNA. The role of specific RNA sequences and structures in promoting the molecular identity of condensates formed for cell polarity and division and by the SARS CoV-2 virus will be discussed.

Synapse formation during neuronal development is critical to establish neural circuits and a nervous system1. Every presynapse builds a core active zone structure where ion channels are clustered and synaptic vesicles are released2. While the composition of active zones is well characterized2,3, how active zone proteins assemble together and recruit synaptic release machinery during development is not clear. Here, we find core active zone scaffold proteins SYD-2/Liprin-α and ELKS-1 phase separate during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation with mutants specifically lacking this activity. These mutant SYD-2 and ELKS-1 proteins remain enriched at synapses, but are defective in active zone assembly and synapse function. The defects are rescued with the introduction of a phase separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid phase scaffold and find it is competent to bind and incorporate downstream active zone components. The fluidity of SYD-2 and ELKS-1 condensates is critical for efficient mixing and incorporation of active zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for synaptic active zone assembly before maturation into a stable final structure.