This allows us to probe also small condensates that would exclude the large particle tracers that are conventionally required for particle tracking experiments 42. The technique is not invasive and can be applied in combination with nanoparticle tracers with size below the optical resolution. DDM probes the microscopic dynamics of the condensates by monitoring fluctuations in the intensity of scattered light over time. Here, we apply differential dynamic microscopy (DDM) to probe the material properties of in vitro models of biomolecular condensates. In this context, several techniques have been developed in soft matter physics, including particle tracking and optical tweezers 17, 36, 37, 40, 41. Techniques capable to probe the dynamics of the systems are ideal to distinguish between liquid-like and gel-/glass-like materials 39. In addition, the molecular factors that modulate these material properties have remained largely unraveled. The assessment of the material properties of condensates, especially of dynamically arrested states, is still very limited in vivo and only a few methods are recently emerging in vitro 36, 37, 38. By contrast, other condensates may require a certain level of rigidity to form a stable structural matrix 24, 32, 33, 34, 35. For instance, for condensates hosting biochemical reactions, fluidity is typically required to recruit client molecules and rapidly release products after processing 31. Understanding the regulation of the material properties after condensate formation and their evolution over time is particularly important. This pathological liquid-to-solid phase transition has been associated with neurodegenerative diseases 26, 27, 28, 29, 30. In some cases, maturation from a liquid-like state into such arrested states has been observed over time 24, 25, potentially leading to the formation of aberrant protein aggregates or amyloids. A variety of material properties ranging from liquid-like to dynamically arrested gel- or glass-like have been reported. Yet, suitable material properties (viscosity, elasticity, surface tension) are likely crucial for the proper physiological function of biomolecular condensates and misregulation of these properties may lead to pathologies 21.īiological condensates contain molecular networks whose formation is mediated by multivalent interactions 6, 22 and can therefore be considered as structured network fluids 23. While a lot of attention has been dedicated to the effect of different factors on the formation and dissolution of biomolecular condensates, mechanisms that control their material properties have remained much less explored. An important feature of phase separating systems is the responsiveness to changes in ionic strength and pH, but also factors like ATP 12, 13, nucleic acids 14, 15, 16, 17, 18, 19, and small molecules 20. These multivalent interactions can be promoted by intrinsically disordered protein sequences known as low-complexity domains (LCDs) 10, by globular protein–protein interactions 8 or by RNA–protein interactions 11. The dynamic formation and dissolution of these biomolecular condensates is governed by a variety of intermolecular interactions 5, which involve multivalency and repetitive sequence patterns 6, 7, 8, 9. In addition to membrane-bound compartments, it is becoming increasingly clear that cells form membraneless organelles by liquid–liquid phase separation (LLPS) of proteins and nucleic acids 1, 2, 3, 4. The ability of cells to form compartments is crucial to coordinate a variety of reactions in space and time.
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