Fleurie M. Kelley

Partitioning of client molecules into biomolecular condensates is critical for regulating the composition and function of condensates. Previous studies suggest that client size limits partitioning. Here, we ask whether large clients, such as macromolecular complexes and nanoparticles, can partition into condensates based on particle-condensate interactions. We seek to discover the fundamental biophysical principles that govern particle inclusion in or exclusion from condensates, using polymer nanoparticles surface-functionalized with biotin or oligonucleotides. Based on our experiments, coarse-grained molecular dynamics simulations, and theory, we conclude that arbitrarily large particles can controllably partition into condensates given sufficiently strong condensate-particle interactions. Remarkably, we also observe that beads with distinct surface chemistries partition orthogonally into immiscible condensates. These findings may provide insights into how various cellular processes are achieved based on partitioning of large clients into biomolecular condensates, and they offer design principles for drug delivery systems that selectively target disease-related condensates.

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.