Surface tension plays a significant role in many functions of biomolecular condensates, from governing the dynamics of droplet coalescence to determining how condensates interact with and deform lipid membranes and biological filaments. To date, however, there is a lack of accurate methods to measure the surface tension of condensates in living cells. Here, we present a high-throughput flicker spectroscopy technique that is able to analyse the thermal fluctuations of the surfaces of tens of thousands of condensates to extract the distribution of surface tensions. Demonstrating this approach on stress granules, we show for the first time that the measured fluctuation spectra cannot be explained by surface tension alone. It is necessary to include an additional energy contribution, which we attribute to an elastic bending rigidity and suggests the presence of structure at the granule-cytoplasm interface. Our data also show that stress granules do not have a spherical base-shape, but fluctuate around a more irregular geometry. Taken together, these results demonstrate quantitatively that the mechanics of stress granules clearly deviate from those expected for simple liquid droplets.

Interfacial tension plays an important role in governing the dynamics of droplet coalescence and determining how condensates interact with and deform lipid membranes and biological filaments. We demonstrate that an interfacial tension-only model is inadequate for describing stress granules in live cells. Harnessing a high-throughput flicker spectroscopy pipeline to analyze the shape fluctuations of tens of thousands of stress granules, we find that the measured fluctuation spectra require an additional contribution, which we attribute to elastic bending deformation. We also show that stress granules have an irregular, nonspherical base shape. These results suggest that stress granules are viscoelastic droplets with a structured interface, rather than simple Newtonian liquids. Furthermore, we observe that the measured interfacial tensions and bending rigidities span a range of several orders of magnitude. Hence, different types of stress granules (and more generally, other biomolecular condensates) can only be differentiated via large-scale surveys.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need.We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

Senescent cells are characterized by an arrest in proliferation. In addition to replicative senescence resulting from telomere exhaustion, sub-lethal genotoxic stress resulting from DNA damage, oncogene activation, mitochondrial dysfunction or reactive metabolites also elicits a senescence phenotype. Senescence is a controlled programme affecting a wide variety of biological processes with some core hallmarks of senescence as well as tissue specific changes. This study presents an integrative multi-omic analysis of proteomic and RNA-seq from proliferating and senescent osteosarcoma cells. This study demonstrates senescence induction in a widely used cell line which can be used as a model system for characterising cancer cell responses to sub-lethal doses of chemotherapeutic agents, and makes available both RNA-seq and proteomic data from proliferating and senescent cells in open access repositories to aid reuse by the community.