Ruedi Meili

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.

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility

Phosphoinositide 3-kinase (PI3K)γ and Dictyostelium PI3K are activated via G protein–coupled receptors through binding to the Gβγ subunit and Ras. However, the mechanistic role(s) of Gβγ and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gβ null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras–guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Cell motility plays an essential role in many biological systems, but precise quantitative knowledge of the biophysical processes involved in cell migration is limited. Better measurements are needed to ultimately build models with predictive capabilities. We present an improved force cytometry method and apply it to the analysis of the dynamics of the chemotactic migration of the amoeboid form of Dictyostelium discoideum. Our explicit calculation of the force field takes into account the finite thickness of the elastic substrate and improves the accuracy and resolution compared with previous methods. This approach enables us to quantitatively study the differences in the mechanics of the migration of wild-type (WT) and mutant cell lines. The time evolution of the strain energy exerted by the migrating cells on their substrate is quasi-periodic and can be used as a simple indicator of the stages of the cell motility cycle. We have found that the mean velocity of migration v and the period of the strain energy T cycle are related through a hyperbolic law v = L/T, where L is a constant step length that remains unchanged in mutants with adhesion or contraction defects. Furthermore, when cells adhere to the substrate, they exert opposing pole forces that are orders of magnitude higher than required to overcome the resistance from their environment.

Rac regulation of chemotaxis and morphogenesis in Dictyostelium

Chemotaxis requires localized F‐actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F‐actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant‐mediated F‐actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k1/2 null cells and wild‐type cells treated with LY294002 exhibit a significantly reduced second peak of RacB activation, which is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F‐actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F‐actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F‐actin polymerization, and chemotaxis in Dictyostelium.

Two Poles and a Compass

Rho GTPases control fundamental aspects of neutrophil chemotaxis: establishment of front and back and orientation toward the chemoattractant. Two reports in this issue show that activated Cdc42 at the leading edge helps orient the cells axis in a signaling complex with G beta gamma, PAK1, and PIX alpha; while Rho, activated via G alpha 13, mediates formation of the uropod, which then interacts by mutual negative feedback with the front to reinforce polarization (Li et al., 2003 [this issue of Cell]; Xu et al., [this issue of Cell]).

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium

BACKGROUND Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility

Amoeboid motility results from pseudopod protrusions and retractions driven by traction forces of cells. We propose that the motor and actin-crosslinking functions of MyoII differentially control the temporal and spatial distribution of the traction forces, and establish mechanistic relationships between these distributions, enabling cells to move.

Dictyostelium: a model for regulated cell movement during morphogenesis

Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces during aggregation.

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility

During chemotactic movement, D. discoideum exhibits step-wise amoeboid motility driven by both contractile axial forces and lateral forces.