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)P(3), at their up-gradient edges; the internal PtdIns(3,4,5)P(3) 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)P(3) 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)P(3) 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)P(3) 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)P(3) and polymerized actin initiates and maintains the asymmetry of intracellular signals responsible for cell polarity and directed motility.

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.

We investigated serotonin signaling in C. elegans as a paradigm for neural regulation of energy balance and found that serotonergic regulation of fat is molecularly distinct from feeding regulation. Serotonergic feeding regulation is mediated by receptors whose functions are not required for fat regulation. Serotonergic fat regulation is dependent on a neurally expressed channel and a G protein-coupled receptor that initiate signaling cascades that ultimately promote lipid breakdown at peripheral sites of fat storage. In turn, intermediates of lipid metabolism generated in the periphery modulate feeding behavior. These findings suggest that, as in mammals, C. elegans feeding behavior is regulated by extrinsic and intrinsic cues. Moreover, obesity and thinness are not solely determined by feeding behavior. Rather, feeding behavior and fat metabolism are coordinated but independent responses of the nervous system to the perception of nutrient availability.

Highlights d aS impacts lipid homeostasis, triggering excess oleic acid (OA) and diglycerides (DG) d Triglycerides and lipid droplets protect against toxicity by sequestering OA and DG d Stearoyl-CoA desaturase (SCD) inhibition rescues aS toxicity and neuron degeneration d SCD inhibition decreases aS inclusions and increases aS multimerization and solubility

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools.