Richard A. Firtel

Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, we show that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis.

Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.

Chemotaxis‐competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein‐coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide‐3 kinase. pkbA null cells exhibit aggregation‐stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.

Signaling pathways controlling cell polarity and chemotaxis.

Many important biological processes, including chemotaxis (directional cell movement up a chemoattractant gradient), require a clearly established cell polarity and the ability of the cell to respond to a directional signal. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways.

The regulation of cell motility and chemotaxis by phospholipid signaling.

Phosphoinositide 3-kinase (PI3K), PTEN and localized phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] play key roles in chemotaxis, regulating cell motility by controlling the actin cytoskeleton in Dictyostelium and mammalian cells. PtdIns(3,4,5)P3, produced by PI3K, acts via diverse downstream signaling components, including the GTPase Rac, Arf-GTPases and the kinase Akt (PKB). It has become increasingly apparent, however, that chemotaxis results from an interplay between the PI3K-PTEN pathway and other parallel pathways in Dictyostelium and mammalian cells. In Dictyostelium, the phospholipase PLA2 acts in concert with PI3K to regulate chemotaxis, whereas phospholipase C (PLC) plays a supporting role in modulating PI3K activity. In adenocarcinoma cells, PLC and the actin regulator cofilin seem to provide the direction-sensing machinery, whereas PI3K might regulate motility.

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cells compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways

Chemoattractant-responsive cells are able to translate a shallow extracellular chemical gradient into a steep intracellular gradient resulting in the localization of F-actin assembly at the front and an actomyosin network at the rear that moves the cell forward. Recent evidence suggests that one of the first asymmetric cellular responses is the localized accumulation of PtdIns(3,4,5)P3, the product of class I phosphoinositide 3-kinase (PI3K) at the site of the new leading edge. The strong accumulation of PtdIns(3,4,5)P3 results from the localized activation of PI3K and also from feedback loops that amplify PtdIns(3,4,5)P3 synthesis at the front and control its degradation at the side and back of cells. These different pathways are temporally and spatially regulated and integrate with other signaling pathways during directional sensing and chemotaxis.

Induction and modulation of cell-type-specific gene expression in dictyostelium

We have identified genes that are expressed preferentially in either prestalk or prespore cells in Dictyostelium. The prestalk mRNAs are detectable at 7.5 hr prior to the completion of cell aggregation, while the prespore mRNAs are not detectable until approximately 15 hr of development. Exogenous cAMP in the absence of sustained cell contact is sufficient to induce prestalk-specific gene expression, while multicellularity is required for the induction of prespore-specific genes. A gene expressed equally in both cell types, which has the same developmental kinetics as the prestalk genes, is induced in shaking culture in the absence of either cAMP or stable cell associations. Dissociation of aggregates results in the rapid loss of prespore- and prestalk-specific mRNAs, and these can be induced to reaccumulate with the addition of cAMP. We conclude that there are substantial differences in the timing and requirements for tissue-specific gene expression in Dictyostelium.

Control of Cell Polarity and Chemotaxis by Akt/PKB and PI3 Kinase through the Regulation of PAKa

We demonstrate that PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, which is required for myosin II assembly. We demonstrate that PI3K and PKB mediate PAKas subcellular localization, PAKas activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKas activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis.

Developmental regulation of a dictyostelium gene encoding a protein homologous to mammalian ras protein

We have cloned, sequenced, and examined the regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras proteins. The Dictyostelium, yeast, and mammalian proteins have homologous N-terminal regions and less conserved C-terminal regions. We have used DNA probes and a polyclonal antibody to examine the differential accumulation of ras RNA and protein through development. The gene encodes two mRNAs (0.9 and 1.2 kb) that are differentially expressed. The 1.2 kb RNA is found in vegetative cells and disappears rapidly upon initiation of development. Later, both RNAs accumulate preferentially in prestalk cells. The level of the Dd-ras protein remains constant until early culmination and then decreases. Like other prestalk genes, Dd-ras can be induced with cAMP in the absence of cell contact. When aggregated cells are dissociated, both mRNAs decrease. Upon addition of cAMP, the 1.2 kb mRNA reaccumulates at a higher level than that in normal developing cells. The presence of the Dd-ras protein in vegetative cells corroborates other reports suggesting a possible function during cell growth. The sustained level of Dd-ras protein in prestalk cells suggests an additional role during differentiation.

Regulation and function of Gα protein subunits in Dictyostelium

Abstract We have examined the developmental regulation and function of two G α protein subunits, G α 1 and G α 2, from Dictyostelium. G α 1 is expressed in vegetative cells through aggregate stages while G α 2 is inducible by cAMP pulses and preferentially expressed in aggregation. Our results suggest that G α 2 encodes the G α protein subunit associated with the cAMP receptor and mediates all known receptor-activated intracellular signal transduction processes, including chemotaxis and gene regulation. G α 1 appears to function in both the cell cycle and development. Overexpression of G α 1 results in large, multinucleated cells that develop abnormally. The central role that these G α proteins play in signal transduction processes and in controlling Dictyostelium development is discussed.