Accurate measurement of the mechanical properties of biomolecular condensates is an essential step in understanding their behaviour within cells. We present FlickerPrint, an open-source Python package to determine the interfacial tension and bending rigidity of thousands of biomolecular condensates using flicker spectroscopy by analysing their shape fluctuations in confocal microscopy images. We detail the workflow used by FlickerPrint to scale up these individual measurements to the population level and the computational requirements to run FlickerPrint. We provide examples of experiments in live cells and in vitro which are suitable for analysis with FlickerPrint as well as scenarios where the package cannot be used. Using these examples, we show that the results obtained from the analysis are robust to changes in the imaging setup, including frame rate. This implementation enables a step-change in the capability to measure two key properties of biomolecular condensates, the interfacial tension and bending rigidity. Moreover, the tools in FlickerPrint are also applicable for analysing other soft, fluctuating bodies, which we demonstrate here using vesicles. ### Competing Interest Statement The authors have declared no competing interest.

Accurate measurement of biomolecular condensates’ mechanical properties is essential to understand their behavior within cells. We present FlickerPrint, an open-source Python package to determine the interfacial tension and bending rigidity of thousands of condensates using flicker spectroscopy by analyzing their shape fluctuations in confocal microscopy images. We detail the workflow and computational requirements of FlickerPrint to scale up these individual measurements to the population level. Examples of experiments in live cells and in vitro that are suitable for analysis with FlickerPrint are provided, as well as scenarios where the package cannot be used. Using these examples, we show that the results obtained are robust to changes in imaging setup, including frame rate. This implementation enables a step change in measurement capability for two key properties of biomolecular condensates: interfacial tension and bending rigidity. Moreover, the tools in FlickerPrint are also applicable for analyzing other soft, fluctuating bodies, demonstrated here using vesicles.

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.

DJ-1 is a redox sensitive protein with a wide range of functions related to oxidative stress protection. Mutations in the park7 gene, which codes for DJ-1 are associated with early onset familial Parkinson's disease and increased astrocytic DJ-1 levels are found in pathologic tissues from idiopathic Parkinson's disease. We have previously established a DJ-1 knockout zebrafish line that developed normally, but with aging the DJ-1 null fish had a lowered level of tyrosine hydroxylase, respiratory mitochondrial failure and a lower body mass. Here we have examined the DJ-1 knockout from the early adult stage and show that loss of DJ-1 results in a progressive, age-dependent increase in both motoric and non-motoric symptoms associated to Parkinson's disease. These changes coincide with changes in mitochondrial and mitochondrial associated proteins. Recent studies have suggested that a decline in NAD+ can contribute to Parkinson's disease and that supplementation of NAD+ precursors may delay disease progression. We found that the brain NAD+/NADH ratio decreased in aging zebrafish but did not correlate with DJ-1 induced altered behavior. Differences were first observed at the late adult stage in which NAD+ and NADPH levels were decreased in DJ-1 knockouts. Considering the experimental power of zebrafish and the development of Parkinson's disease-related symptoms in the DJ-1 null fish, this model can serve as a useful tool both to understand the progression of the disease and the effect of suggested treatments.

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.

Coacervates droplets have long been considered as potential protocells to mimic living cells. However, these droplets lack a membrane and are prone to coalescence, limiting their ability to survive, interact, and organize into higher-order assemblies. This work shows that tyrosine-rich peptide conjugates can undergo liquid-liquid phase separation in a well-defined pH window and transform into stable membrane-enclosed protocells by enzymatic oxidation and cross-linking at the liquid-liquid interface. The oxidation of the tyrosine-rich peptides into dityrosine creates a semipermeable, flexible membrane around the coacervates with tunable thickness, which displays strong intrinsic fluorescence, and stabilizes the coacervate protocells against coalescence. The membranes have an effective molecular weight cut-off of 2.5 kDa, as determined from the partitioning of small dyes and labeled peptides, RNA, and polymers into the membrane-enclosed coacervate protocells. Flicker spectroscopy reveals a membrane bending rigidity of only 0.1k(B)T, which is substantially lower than phospholipid bilayers despite a larger membrane thickness. Finally, it is shown that enzymes can be stably encapsulated inside the protocells and be supplied with substrates from outside, which opens the way for these membrane-bound compartments to be used as molecularly crowded artificial cells capable of communication or as a vehicle for drug delivery.

For particles confined to two dimensions, any curvature of the surface affects the structural, kinetic and thermodynamic properties of the system. If the curvature is non-uniform, an even richer range of behaviours can emerge. Using a combination of bespoke Monte Carlo, molecular dynamics and basin-hopping methods, we show that the stable states of attractive colloids confined to non-uniformly curved surfaces are distinguished not only by the phase of matter but also by their location on the surface. Consequently, the transitions between these states involve cooperative migration of the entire colloidal assembly. We demonstrate these phenomena on toroidal and sinusoidal surfaces for model colloids with different ranges of interactions as described by the Morse potential. In all cases, the behaviour can be rationalised in terms of three universal considerations: cluster perimeter, stress, and the packing of next-nearest neighbours.

We develop a theoretical framework for understanding dynamic morphologies and stability of droplet interface bilayers (DIBs), accounting for lipid kinetics in the monolayers and bilayer, and droplet evaporation due to imbalance between osmotic and Laplace pressures. Our theory quantitatively describes distinct pathways observed in experiments when DIBs become unstable. We find that when the timescale for lipid desorption is slow compared to droplet evaporation, the lipid bilayer will grow and the droplets approach a hemispherical shape. In contrast, when lipid desorption is fast, the bilayer area will shrink and the droplets eventually detach. Our model also suggests there is a critical size below which DIBs can become unstable, which may explain experimental difficulties in miniaturizing the DIB platform.

By combining Monte Carlo simulations and analytical models, we demonstrate and explain how the gas-to-liquid phase transition of colloidal systems confined to a spherical surface depends on the curvature and size of the surface, and on the choice of thermodynamic ensemble. We find that the geometry of the surface affects the shape of the free energy profile and the size of the critical nucleus by altering the perimeter-area ratio of isotropic clusters. Confinement to a smaller spherical surface results in both a lower nucleation barrier and a smaller critical nucleus size. Furthermore, the liquid domain does not grow indefinitely on a sphere. Saturation of the liquid density in the grand canonical ensemble and the depletion of the gas phase in the canonical ensemble lead to a minimum in the free energy profile, with a sharp increase in free energy for additional growth beyond this minimum. [GRAPHICS] .