Cells are organized on length scales ranging from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. Here we show that interactions between diverse, synthetic multivalent macromolecules (including multi-domain proteins and RNA) produce sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its established biological partners Nck and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology.

SUMMARY Members of the Wiskott-Aldrich Syndrome Protein (WASP) family control actin dynamics in eukaryotic cells through stimulating the actin nucleating activity of the Arp2/3 complex. The prevailing paradigm for WASP regulation invokes allosteric relief of autoinhibition by diverse upstream activators. Here we demonstrate an additional level of regulation that is superimposed upon allostery: dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly by this system. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3 domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between different family members. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott-Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.actin assembly ͉ microbial pathogenesis ͉ virulence ͉ stress fibers ͉ WH2 domains

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected by bacteria into eukaryotic host cells. VopL contains three N-terminal WiskottAldrich Homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own, and requires the VCD for activity. The VCD forms a Ushaped dimer in the crystal, which is stabilized by a terminal coiled-coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model where VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.Actin cytoskeletal dynamics are important in numerous cellular processes, including migration, division and maintenance of morphology [1][2][3] . These functions all require rapid assembly of new actin filaments de novo from actin monomers. However, actin assembly is intrinsically slow, due to kinetic and thermodynamic barriers to forming the actin dimers and trimers needed to initiate filament growth 4 . Cells have therefore developed specialized factors to stabilize these actin nuclei and thus catalyze filament assembly 4 . Three classes of so-called actin nucleation factors have been identified: Arp2/3 complex, formin proteins and WASP Homology domain 2 (WH2)-based nucleators [5][6][7][8][9][10][11] . Arp2/3 complex binds to the side of an existing filament and initiates growth of a new filament from its actin related protein 2 (Arp2) and Arp3 subunits 12,13 . Alignment of the actin homologs Arp2 and Arp3 to resemble Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript the first two monomers at the base of an actin filament is believed to be a key aspect of Arp2/3-mediated nucleation 14 . Formin proteins nucleate linear filaments through a conserved formin homology 2 (FH2) domain, which is also thought to organize three actin monomers into a structure that resembles the base of an actin filament 15,16 . After nucleation, formins remain associated with the growing filament barbed end as new monomers are added 5 . Several mechanisms have been proposed to account for this processive activity [15][16][17][18] . The WH2-based actin nucleators have been identified most recently, and include Spire, Corbon blue (Cobl) and Leiomodin (Lmod) [7][8][9]11 . These proteins all contain a series of tandem WH2 motifs, each capable of binding an actin monomer. These proteins are also thought to act through organization of multiple actin monomers into a stable structure...