The low complexity domain of the RNA-binding protein FUS (FUS LC) mediates liquid-liquid phase separation (LLPS), but interactions between the repetitive SYGQ-rich sequence of FUS LC that stabilize the liquid phase are not known in detail. By combining NMR and Raman spectroscopy, mutagenesis, and molecular simulation, we demonstrate that heterogeneous interactions involving all residue types underlie LLPS of human FUS LC. We find no evidence that FUS LC adopts conformations with traditional secondary structure elements in the condensed phase, rather it maintains conformational heterogeneity. We show that hydrogen bonding, π/sp2 and hydrophobic interactions all contribute to stabilizing LLPS of FUS LC. In addition to contributions from tyrosine residues, we find that glutamine residues participate in contacts leading to LLPS of FUS LC. These results support a model in which FUS LC forms dynamic, multivalent interactions via multiple residue types and remains disordered in the densely packed liquid phase.

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes [1][2][3] . Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension 4-8 . Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.Monomers of actin assemble into polar filaments that are organized by various actin-binding proteins into branched, bundled and/or crosslinked networks essential for basic cellular functions 1 . In crawling cells, growth of actin filament networks characterized by a dendritic architecture-highly branched structures with short filaments (~0.1-1 µm) oriented in the direction of migration-generates force at the cell periphery for membrane protrusions 1,9,10 .Actin filaments, as well as other biological and synthetic polymers, are categorized by the relationship between their persistence length L p and contour length L c . The persistence length is defined as the average length over which the filament orientation changes due to thermal fluctuations, and the contour length is the length of the completely extended filament. For flexible polymers (L c ≫ L p ) the resistance to extension and compression is determined by the conformational entropy of the chain and is described as entropic elasticity. Flexible polymers exhibit stress stiffening near full extension because there is ultimately only one fully extended conformation, assuming inextensibility 11 . For stiff polymers (L c ≪ L p ) resistance to extension, bending and compression is due to straining of molecular links from equilibrium, which is quantified by the bending modulus κ and ©2007 Nature Publishing GroupCorrespondence and requests for materials should be addressed to D.A.F. (fletch@berkeley.edu). These authors contributed equally to this work.Supplementary Information is linked to...

In nature, aqueous solutions often move collectively along solid surfaces (for example, raindrops falling on the ground and rivers flowing through riverbeds). However, the influence of such motion on water-surface interfacial chemistry is unclear. In this work, we combine surface-specific sum frequency generation spectroscopy and microfluidics to show that at immersed calcium fluoride and fused silica surfaces, flow leads to a reversible modification of the surface charge and subsequent realignment of the interfacial water molecules. Obtaining equivalent effects under static conditions requires a substantial change in bulk solution pH (up to 2 pH units), demonstrating the coupling between flow and chemistry. These marked flow-induced variations in interfacial chemistry should substantially affect our understanding and modeling of chemical processes at immersed surfaces.

We demonstrate 3D super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) let us physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples 8-fold thicker than previously demonstrated with SIM. We imaged a variety of samples at one 2D image per second, at resolutions down to 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths greater than 45 μm. We also captured dynamic changes in the zebrafish lateral line primordium and observed the interactions between myosin IIA and F-actin in cells encapsulated within collagen gels, obtaining two-color 4D super-resolution datasets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with widefield microscopes.