Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.

Protein condensates are complex fluids that can change their material properties with time. However, an appropriate rheological description of these fluids remains missing. We characterize the time-dependent material properties of in vitro protein condensates using laser tweezer–based active and microbead-based passive rheology. For different proteins, the condensates behave at all ages as viscoelastic Maxwell fluids. Their viscosity strongly increases with age while their elastic modulus varies weakly. No significant differences in structure were seen by electron microscopy at early and late ages. We conclude that protein condensates can be soft glassy materials that we call Maxwell glasses with age-dependent material properties. We discuss possible advantages of glassy behavior for modulation of cellular biochemistry.

P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory we show that in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development, by using RNA competition to regulate local phase separation.

Collagen is the main structural and load-bearing element of various connective tissues, where it forms the extracellular matrix that supports cells. It has long been known that collagenous tissues exhibit a highly nonlinear stress-strain relationship, although the origins of this nonlinearity remain unknown. Here, we show that the nonlinear stiffening of reconstituted type I collagen networks is controlled by the applied stress and that the network stiffness becomes surprisingly insensitive to network concentration. We demonstrate how a simple model for networks of elastic fibers can quantitatively account for the mechanics of reconstituted collagen networks. Our model points to the important role of normal stresses in determining the nonlinear shear elastic response, which can explain the approximate exponential relationship between stress and strain reported for collagenous tissues. This further suggests principles for the design of synthetic fiber networks with collagen-like properties, as well as a mechanism for the control of the mechanics of such networks.collagen networks | nonlinear elasticity | normal stress | tissue mechanics C ollagen type I is the most abundant protein in mammals where it serves as the primary component of many load-bearing tissues, including skin, ligaments, tendons, and bone. Networks of collagen type I fibers exhibit mechanical properties that are unmatched by manmade materials. A hallmark of collagen and collagenous tissues is a dramatic increase in stiffness when strained. Qualitatively, this property of strain stiffening is shared by many other biopolymers, including intracellular cytoskeletal networks of actin and intermediate filaments (1-5). On closer inspection, however, collagen stands out from the rest: it has been shown that collagenous tissues exhibit a regime in which the stress is approximately exponential in the applied strain (6). The origins of this nonlinearity are still not known (7,8), and existing models for biopolymer networks cannot account quantitatively for collagen. In particular, it is unknown whether the nonlinear mechanical response of collagen originates at the level of the individual fibers (1, 3, 9, 10) or arises from nonaffine network deformations as suggested by numerical simulations (11-17).Here, we present both experimental results on reconstituted collagen networks, as well as a model that quantitatively captures the observed nonlinear mechanics. Our model is a minimal one, of random networks of elastic fibers possessing only bending and stretching elasticity. This model can account for our striking experimental observation that the stiffness of collagen becomes independent of protein concentration in the nonlinear elastic regime, over a range of concentrations and applied shear stress. Our model highlights the importance of local network geometry in determining the strain threshold for the onset of nonlinear mechanics, which can account for the concentration independence of this threshold that is observed for collagen (8,17), in strong contrast...

Blood clots perform an essential mechanical task, yet the mechanical behavior of fibrin fibers, which form the structural framework of a clot, is largely unknown. By using combined atomic force-fluorescence microscopy, we determined the elastic limit and extensibility of individual fibers. Fibrin fibers can be strained 180% (2.8-fold extension) without sustaining permanent lengthening, and they can be strained up to 525% (average 330%) before rupturing. This is the largest extensibility observed for protein fibers. The data imply that fibrin monomers must be able to undergo sizeable, reversible structural changes and that deformations in clots can be accommodated by individual fiber stretching.

We show that the nonlinear mechanical response of networks formed from un-cross-linked fibrin or collagen type I continually changes in response to repeated large-strain loading. We demonstrate that this dynamic evolution of the mechanical response arises from a shift of a characteristic nonlinear stress-strain relationship to higher strains. Therefore, the imposed loading does not weaken the underlying matrices but instead delays the occurrence of the strain stiffening. Using confocal microscopy, we present direct evidence that this behavior results from persistent lengthening of individual fibers caused by an interplay between fiber stretching and fiber buckling when the networks are repeatedly strained. Moreover, we show that covalent cross-linking of fibrin or collagen inhibits the shift of the nonlinear material response, suggesting that the molecular origin of individual fiber lengthening may be slip of monomers within the fibers. Thus, a fibrous architecture in combination with constituents that exhibit internal plasticity creates a material whose mechanical response adapts to external loading conditions. This design principle may be useful to engineer novel materials with this capability.ECM | nonlinear rheology | factor XIII | blood clot N etworks of stiff biopolymer fibers are a major component of the structural architecture of multicellular organisms; their unique material properties provide rigidity and protect structural integrity. These networks are particularly important in the extracellular matrix (ECM) where they provide mechanical support to living cells and form many of the load-carrying structures in the body. One important example is fibrin, which forms the underlying scaffold of blood clots and the provisional matrix (1). Another important example is collagen type I, the major structural constituent of all connective tissue, tendons, ligaments, and bone (2). Because the in vivo structure of these fiber networks is so complex, investigations of in vitro networks of both proteins have been used to explore their structure and unique mechanical properties, and to elucidate their underlying design principles. Interestingly, fibrin and collagen exhibit many similar features: Both proteins self-assemble into thick, hierarchically ordered, rather stiff fibers through electrostatic and hydrophobic interactions (3, 4); these fibers associate into sparse, 3D networks that possess unusual mechanical properties not seen in synthetic polymers. In both cases, these networks display highly nonlinear mechanics and stiffen significantly as the strain increases (5-8). In addition, they are both also viscoelastic: They partially store elastic energy and partially relax internal stress through dissipative processes (9-12). All of these properties are delicately influenced by the structure of the networks, by the molecular interactions between monomers, and by the addition of covalent cross-links (7,13,14). This creates a delicate interplay between the viscoelastic and nonlinear mechanical properties of these ne...

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young's modulus, E) and breaking strain (also called rupture strain or extensibility, epsilon(max)) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young's modulus on the order of a few Gigapascal and are not very extensible (epsilon(max) < 20%). They also display a very regular arrangement of their monomeric units. Soft biological protein fibers have a Young's modulus on the order of a few Megapascal and are very extensible (epsilon(max) > 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an alpha-helix to beta-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the gamma-chain.

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquid-like phases. These protein condensates serve as simple models for the investigation of the more complex membrane-less organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties. arXiv:1809.09832v2 [cond-mat.soft] 6 Dec 2018 arXiv:1809.09832v2 [cond-mat.soft]