Two distinct functions for PI3-kinases in macropinocytosis.

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

RasG signaling is important for optimal folate chemotaxis in Dictyostelium.

BACKGROUND: Signaling pathways linking receptor activation to actin reorganization and pseudopod dynamics during chemotaxis are arranged in complex networks. Dictyostelium discoideum has proven to be an excellent model system for studying these networks and a body of evidence has indicated that RasG and RasC, members of the Ras GTPase subfamily function as key chemotaxis regulators. However, recent evidence has been presented indicating that Ras signaling is not important for Dictyostelium chemotaxis. In this study, we have reexamined the role of Ras proteins in folate chemotaxis and then, having re-established the importance of Ras for this process, identified the parts of the RasG protein molecule that are involved. RESULTS: A direct comparison of folate chemotaxis methodologies revealed that rasG-C- cells grown in association with a bacterial food source were capable of positive chemotaxis, only when their initial position was comparatively close to the folate source. In contrast, cells grown in axenic medium orientate randomly regardless of their distance to the micropipette. Folate chemotaxis is restored in rasG-C- cells by exogenous expression of protein chimeras containing either N- or C- terminal halves of the RasG protein. CONCLUSIONS: Conflicting data regarding the importance of Ras to Dictyostelium chemotaxis were the result of differing experimental methodologies. Both axenic and bacterially grown cells require RasG for optimal folate chemotaxis, particularly in weak gradients. In strong gradients, the requirement for RasG is relaxed, but only in bacterially grown cells. Both N- and C- terminal portions of the RasG protein are important for folate chemotaxis, suggesting that there are functionally important amino acids outside the well established switch I and switch II interaction surfaces.

Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division.

BACKGROUND: Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans. RESULTS: In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC. CONCLUSIONS: Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.

Determinants of RasC specificity during Dictyostelium aggregation.

RasC is required for optimum activation of adenylyl cyclase A and for aggregate stream formation during the early differentiation of Dictyostelium discoideum. RasG is unable to substitute for this requirement despite its sequence similarity to RasC. A critical question is which amino acids in RasC are required for its specific function. Each of the amino acids within the switch 1 and 2 domains in the N-terminal portion of RasG was changed to the corresponding amino acid from RasC, and the ability of the mutated RasG protein to reverse the phenotype of rasC(-) cells was determined. Only the change from aspartate at position 30 of RasG to alanine (the equivalent position 31 in RasC) resulted in a significant increase in adenylyl cyclase A activation and a partial reversal of the aggregation-deficient phenotype of rasC(-) cells. All other single amino acid changes were without effect. Expression of a chimeric protein, RasG(1-77)-RasC(79-189), also resulted in a partial reversal of the rasC(-) cell phenotype, indicating the importance of the C-terminal portion of RasC. Furthermore, expression of the chimeric protein, with alanine changed to aspartate (RasG(1-77(D30A))-RasC(79-189)), resulted in a full rescue the rasC(-) aggregation-deficient phenotype. Finally, the expression of either a mutated RasC, with the aspartate 31 replaced by alanine, or the chimeric protein, RasC(1-78)-RasG(78-189), only generated a partial rescue. These results emphasize the importance of both the single amino acid at position 31 and the C-terminal sequence for the specific function of RasC during Dictyostelium aggregation.

PIP3-independent activation of TorC2 and PKB at the cell's leading edge mediates chemotaxis.

BACKGROUND: Studies show that high phosphotidylinositol 3,4,5-trisphosphate (PIP(3)) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of protein kinase Bs (PKBs). However, chemotaxis can still occur in the absence of PIP(3), and the identities of the PIP(3)-independent pathways remain unknown. RESULTS: Here, we outline a PIP(3)-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two Ras GEFs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events are restricted to the cell's leading edge even in the absence of PIP(3). Activation of TorC2 depends on heterotrimeric G protein function and intermediate G proteins, including Ras GTPases. CONCLUSIONS: The data lead to a model where cytosolic TorC2, encountering locally activated small G protein(s) at the leading edge of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration.

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum.

On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development. Both chemotaxis and ACA activation were reduced in the rasG- cells, but the effect on chemotaxis was more pronounced. When the responses of rasG- cells to cAMP were compared with the responses of rasC- and rasC- rasG- strains, generated in otherwise isogenic backgrounds, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Because the loss of either of the two Ras proteins alone did not result in a total loss of signal output down either of the branches of the cAMP signal-response pathway, there appears to be some overlap of function.

The small GTPases Ras and Rap1 bind to and control TORC2 activity.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

Developmental lineage priming in Dictyostelium by heterogeneous Ras activation.

In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.

A Rap/phosphatidylinositol 3-kinase pathway controls pseudopod formation [corrected].

GbpD, a Dictyostelium discoideum guanine exchange factor specific for Rap1, has been implicated in adhesion, cell polarity, and chemotaxis. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. Phg2, a serine/threonine-specific kinase, mediates Rap1-regulated cell-substrate adhesion, but not cell polarity or chemotaxis. In this study we demonstrate that overexpression of GbpD in pi3k1/2-null cells does not induce the adhesion and cell morphology phenotype. Furthermore we show that Rap1 directly binds to the Ras binding domain of PI3K, and overexpression of GbpD leads to strongly enhanced PIP3 levels. Consistently, upon overexpression of the PIP3-degradating enzyme PTEN in GbpD-overexpressing cells, the strong adhesion and cell morphology phenotype is largely lost. These results indicate that a GbpD/Rap/PI3K pathway helps control pseudopod formation and cell polarity. As in Rap-regulated pseudopod formation in Dictyostelium, mammalian Rap and PI3K are essential for determining neuronal polarity, suggesting that the Rap/PI3K pathway is a conserved module regulating the establishment of cell polarity.

Regulation of Rap1 activity is required for differential adhesion, cell-type patterning and morphogenesis in Dictyostelium.

Regulated cell adhesion and motility have important roles during growth, development and tissue homeostasis. Consequently, great efforts have been made to identify genes that control these processes. One candidate is Rap1, as it has been implicated in the regulation of adhesion and motility in cell culture. To further study the role of Rap1 during multicellular development, we generated a mutant in a potential Rap1 GTPase activating protein (RapGAPB) in Dictyostelium. rapGAPB(-) cells have increased levels of active Rap1 compared with wild-type cells, indicating that RapGAPB regulates Rap1 activity. Furthermore, rapGAPB(-) cells exhibit hallmark phenotypes of other known mutants with hyperactivated Rap1, including increased substrate adhesion and abnormal F-actin distribution. However, unlike these other mutants, rapGAPB(-) cells do not exhibit impaired motility or chemotaxis, indicating that RapGAPB might only regulate specific roles of Rap1. Importantly, we also found that RapGAPB regulates Rap1 activity during multicellular development and is required for normal morphogenesis. First, streams of aggregating rapGAPB(-) cells break up as a result of decreased cell-cell adhesion. Second, rapGAPB(-) cells exhibit cell-autonomous defects in prestalk cell patterning. Using cell-type-specific markers, we demonstrate that RapGAPB is required for the correct sorting behaviour of different cell types. Finally, we show that inactivation of RapGAPB affects prestalk and prespore cell adhesion. We therefore propose that a possible mechanism for RapGAPB-regulated cell sorting is through differential adhesion.

Rap1 activation in response to cAMP occurs downstream of ras activation during Dictyostelium aggregation.

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.