Cellular surfaces as regulators of biomolecular condensation*

All cells depend on subcellular compartments to organize diverse biochemical processes. Membraneless biomolecular condensates compartmentalize cells by enriching certain proteins and nucleic acids within assemblies that can display properties of liquid-like droplets. A key requirement for condensate function is the dynamic regulation of size, which impacts molecular exchange, reaction efficiency, and environmental responsiveness. However, condensates reconstituted from minimal components in a test tube often vastly exceed the sizes of native condensates in cells. The biophysical mechanisms that control condensate size are incompletely understood. Many condensates associate with biological “surfaces,” including (a) two-dimensional lipid bilayer membranes and (b) one-dimensional polymers of long noncoding RNA. In this talk, I will share two stories of how membrane and polymer surfaces control condensate size. In the first story, I found that attachment to membranes controls the sizes of condensates composed of the protein Whi3. Specifically, Whi3 tends to form large droplets in vitro, but Whi3 condensates in cells appear as small puncta. I discovered that Whi3 condensates are attached to endomembranes in cells. Recruiting Whi3 to membranes in vitro restricts condensate growth substantially, resulting in punctate condensates that resemble native assemblies. The slow growth was due to the slower diffusion of membrane-bound molecules compared to solution. In the second story, I found that a long noncoding RNA called NEAT1 controls the sizes of nuclear paraspeckles. NEAT1 contains binding sites for multiple proteins, including FUS and NONO. While FUS tends to form large droplets with NEAT1 in vitro, paraspeckles appear as small puncta with regular sizes of ~360 nm. I discovered that FUS-NEAT1 condensates are smaller in the presence of NONO, suggesting that NONO opposes FUS condensation by competing for binding to NEAT1. These data indicate that NEAT1 controls paraspeckle size by tuning the recruitment of competing proteins. In both stories, biological surfaces control condensate sizes without active, energy-consuming processes. Given that many condensates associate with surfaces throughout the cell, these findings reveal broadly-applicable mechanisms of size regulation.