Henry R. Bourne

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-γ and PTEN

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol–phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-γ (PI(3)Kγ) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kγ and PTEN control electrotaxis.

How receptors talk to trimeric G proteins

Stimulated by hormones and sensory stimuli, serpentine receptors promote the release of GDP that is bound to the alpha subunit of trimeric G proteins and its replacement by GTP. Recent investigations have begun to define the sizes, shapes, and relative orientations of receptors and G proteins, the surfaces through which they interact with one another, and conformational changes in both sets of molecules that underlie receptor-catalyzed guanine-nucleotide exchange.

A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity.

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P3. This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium discoideum and fibroblasts. However, the mechanisms that regulate PtdInsP3 polarization are not well understood. Using a cationic lipid shuttling system, we have delivered exogenous PtdInsP3 to neutrophils. Exogenous PtdInsP3 elicits accumulation of endogenous PtdInsP3 in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI(3)Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP3 polarity in response to both chemoattractant and to exogenous PtdInsP3; it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP3 and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis.

Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils

In gradients of external chemo-attractant, mammalian neutrophilic leukocytes (neutrophils) and Dictyostelium discoideum amoebae adopt a polarized morphology and selectively accumulate lipid products of phosphatidylinositol-3-OH kinases (PI(3)Ks), including PtdIns(3,4,5)P3, at their up-gradient edges; the internal PtdIns(3,4,5)P3 gradient substantially exceeds that of the external attractant. An accompanying report presents evidence for a positive feedback loop that amplifies the gradient of internal signal: PtdIns(3,4,5)P3 at the leading edge stimulates its own accumulation by inducing activation of one or more Rho GTPases (Rac, Cdc42, and/or Rho), which in turn increase PtdIns(3,4,5)P3 accumulation. Here we show that interruption of this feedback by treatment with PI(3)K inhibitors reduces the size and stability of pseudopods and causes cells to migrate in jerky trajectories that deviate more from the up-gradient direction than do those of controls. Moreover, amplification of the internal PtdIns(3,4,5)P3 gradient is markedly impaired by latrunculin or jasplakinolide, toxins that inhibit polymerization or depolymerization of actin, respectively. Thus reciprocal interplay between PtdIns(3,4,5)P3 and polymerized actin initiates and maintains the asymmetry of intracellular signals responsible for cell polarity and directed motility.

Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

Leukocytes navigate by compass: roles of PI3Kγ and its lipid products

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.

Activation and depalmitoylation of Gsα

Abstract [ 3 H]palmitate attached to mutationally activated α s ( α s -R201C) turns over rapidly, compared with palmitate linked to normal α s (t12 ≈ 2 min versus 90 min); although α s -R201C (unlike normal α s ) is predominantly found in the cytosol, [ 3 H]palmitate is linked only to the membrane-bound pool of α s , normal or mutant. Similarly, activation of wild-type α s by isoproterenol, a β-adrenoceptor agonist that also induces membrane-to-cytosol translocation of α s , dramatically accelerates depalmitoylation of α s . Thus, activation-induced removal of palmitate provides an explanation for activation-induced shifts of α s to the cytosol. Regulated palmitoylation cycles provide a potential general mechanism for controlling reversible changes in the cellular location and activity of a protein.

Molecular machines integrate coincident synaptic signals

Every synaptic event represents the summed behavior of populations of proteins that receive signals and relay them, amplified and transformed, to other proteins. Encoded by thousands of different genes, these protein signaling machines fall into a small number of families-the protein kinases, phosphatases, GTPases, ligand- and voltage-gated ion channels, several varieties of cell surface receptors, and a few others. Because each family is characterized by a shared design and a common molecular mechanism, we can realistically hope to understand how these machines work

Spatial control of actin polymerization during neutrophil chemotaxis

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

G-protein diseases furnish a model for the turn-on switch

How does a trimeric G protein on the inside of a cell membrane respond to activation by a transmembrane receptor? G-protein mutations in patients with hypertension and inherited endocrine disorders enhance or block signals from stimulated receptors. In combination with three-dimensional crystal structures and results from biochemical experiments, the phenotypes produced by these mutations suggest a model for the molecular activation mechanism that relays hormonal and sensory signals transmitted by many transmembrane receptors.