Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation.

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state

Formin-dependent TGF-β signaling for epithelial to mesenchymal transition.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-β. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-β receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-β. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-β signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ

A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

Increased intracellular H+ efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1−) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1− cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI(3,4,5)P3 production and for attaining a polarized phenotype

Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration

Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments

The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical “or gate” capable integrating diverse upstream signals

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions

Positive feedback between Cdc42 activity and H + efflux by the Na-H exchanger NHE1 for polarity of migrating cells

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H+ efflux by Na-H+ exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H+ efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H+ efflux by NHE1 is not necessary for release of Cdc42–guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5–bisphosphate is pH dependent, suggesting a mechanism for how H+ efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells

Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics

The glycolytic enzyme phosphofructokinase-1 assembles into filaments.

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells