Peter N. Devreotes

Tumor Suppressor PTEN Mediates Sensing of Chemoattractant Gradients

Shallow gradients of chemoattractants, sensed by G protein-linked signaling pathways, elicit localized binding of PH domains specific for PI(3,4,5)P3 at sites on the membrane where rearrangements of the cytoskeleton and pseudopod extension occur. Disruption of the PI 3-phosphatase, PTEN, in Dictyostelium discoideum dramatically prolonged and broadened the PH domain relocation and actin polymerization responses, causing the cells lacking PTEN to follow a circuitous route toward the attractant. Exogenously expressed PTEN-GFP localized to the surface membrane at the rear of the cell. Membrane localization required a putative PI(4,5)P2 binding motif and was required for chemotaxis. These results suggest that specific phosphoinositides direct actin polymerization to the cells leading edge and regulation of PTEN through a feedback loop plays a critical role in gradient sensing and directional migration.

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.

G PROTEIN SIGNALING EVENTS ARE ACTIVATED AT THE LEADING EDGE OF CHEMOTACTIC CELLS

Directional sensing by eukaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH domain-containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient, the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes.

Chemotaxis: signalling the way forward

During random locomotion, human neutrophils and Dictyostelium discoideum amoebae repeatedly extend and retract cytoplasmic processes. During directed cell migration — chemotaxis — these pseudopodia form predominantly at the leading edge in response to the local accumulation of certain signalling molecules. Concurrent changes in actin and myosin enable the cell to move towards the stimulus. Recent studies are beginning to identify an intricate network of signalling molecules that mediate these processes, and how these molecules become localized in the cell is now becoming clear.

Heterotrimeric G-protein

Receptor mediated activation of heterotrimeric G-proteins is visualized in living cells by monitoring fluorescence resonance energy transfer (FRET) between subunits of a G protein fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociates and reassociates upon addition and removal of cognate ligand. Energy transfer pairs of G-proteins enables direct in situ detection and have applications for drug screening and GPCR de-orphaning.

Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.

Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.

Temporal and Spatial Regulation of Chemotaxis

The ability to sense and respond to shallow gradients of extracellular signals is remarkably similar in Dictyostelium discoideum amoebae and mammalian leukocytes. Chemoattractant receptors and G proteins are fairly evenly distributed along the cell surface. Receptor occupancy generates local excitatory and global inhibitory processes that balance to control the chemotactic response. Uniform stimuli transiently recruit PI3Ks to, and release PTEN from, the plasma membrane, while gradients of chemoattractant cause the two enzymes to bind to the membrane at the front and back of the cell, respectively. Interference with PI3Ks alters chemotaxis, and disruption of PTEN broadens PI localization and actin polymerization in parallel. Thus, counteracting signals from the upstream elements of the pathway converge to regulate the key enzymes of PI metabolism, localize these lipids, and direct pseudopod formation.

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in dictyostelium development

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Navigating through models of chemotaxis

Chemotaxis in eukaryotic cells involves the coordination of several related but separable processes: motility, polarization, and gradient sensing. Mathematical models that have been proposed to explain chemotaxis typically focus on only one of these processes. We summarize the strengths and weaknesses of the models and point out the need for an integrated model.

A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN

The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP2) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP2 and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN1–351, bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN352–403. The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP2 binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.