Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein.

Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.

The WAVE regulatory complex links diverse receptors to the actin cytoskeleton.

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.

The bacterial effector VopL organizes actin into filament-like structures.

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.

Incorporation of cofilin into rods depends on disulfide intermolecular bonds: implications for actin regulation and neurodegenerative disease.

Rod-shaped aggregates ("rods"), containing equimolar actin and the actin dynamizing protein cofilin, appear in neurons following a wide variety of potentially oxidative stress: simulated microischemia, cofilin overexpression, and exposure to peroxide, excess glutamate, or the dimer/trimer forms of amyloid-ß peptide (Aßd/t), the most synaptotoxic Aß species. These rods are initially reversible and neuroprotective, but if they persist in neurites, the synapses degenerate without neurons dying. Herein we report evidence that rod formation depends on the generation of intermolecular disulfide bonds in cofilin. Of four Cys-to-Ala cofilin mutations expressed in rat E18 hippocampal neurons, only the mutant incapable of forming intermolecular bonds (CC39,147AA) has significantly reduced ability to incorporate into rods. Rod regions show unusually high oxidation levels. Rods, isolated from stressed neurons, contain dithiothreitol-sensitive multimeric forms of cofilin, predominantly dimer. Oligomerization of cofilin in cells represents one more mechanism for regulating the actin dynamizing activity of cofilin and probably underlies synaptic loss.

Direct redox regulation of F-actin assembly and disassembly by Mical.

Different types of cell behavior, including growth, motility, and navigation, require actin proteins to assemble into filaments. Here, we describe a biochemical process that was able to disassemble actin filaments and limit their reassembly. Actin was a specific substrate of the multidomain oxidation-reduction enzyme, Mical, a poorly understood actin disassembly factor that directly responds to Semaphorin/Plexin extracellular repulsive cues. Actin filament subunits were directly modified by Mical on their conserved pointed-end, which is critical for filament assembly. Mical posttranslationally oxidized the methionine 44 residue within the D-loop of actin, simultaneously severing filaments and decreasing polymerization. This mechanism underlying actin cytoskeletal collapse may have broad physiological and pathological ramifications.

Growth cone-like waves transport actin and promote axonogenesis and neurite branching.

Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone-like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in nonwave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP-actin and photoconversion of Dendra-actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics.

Actin-binding proteins take the reins in growth cones.

Higher-order actin-based networks (actin superstructures) are important for growth-cone motility and guidance. Principles for generating, organizing and remodelling actin superstructures have emerged from recent findings in cell-free systems, non-neuronal cells and growth cones. This Review examines how actin superstructures are initiated de novo at the leading-edge membrane and how the spontaneous organization of actin superstructures is driven by ensembles of actin-binding proteins. How the regulation of actin-binding proteins can affect growth-cone turning and axonal regeneration is also discussed.