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Richard A Firtel

Publications and source records attributed to Richard A Firtel.

At least 19 recordsLinked to original sources
PubMed2007

Big roles for small GTPases in the control of directed cell movement.

Small GTPases are involved in the control of diverse cellular behaviours, including cellular growth, differentiation and motility. In addition, recent studies have revealed new roles for small GTPases in the regulation of eukaryotic chemotaxis. Efficient chemotaxis results from co-ordinated chemoattractant gradient sensing, cell polarization and cellular motility, and accumulating data suggest that small GTPase signalling plays a central role in each of these processes as well as in signal relay. The present review summarizes these recent findings, which shed light on the molecular mechanisms by which small GTPases control directed cell migration.

Actins↗
PubMed2006

Feedback signaling controls leading-edge formation during chemotaxis.

Chemotactic cells translate shallow chemoattractant gradients into a highly polarized intracellular response that includes the localized production of PI(3,4,5)P(3) on the side of the cell facing the highest chemoattractant concentration. Research over the past decade began to uncover the molecular mechanisms involved in this localized signal amplification controlling the leading edge of chemotaxing cells. These mechanisms have been shown to involve multiple positive feedback loops, in which the PI(3,4,5)P(3) signal amplifies itself independently of the original stimulus, as well as inhibitory signals that restrict PI(3,4,5)P(3) to the leading edge, thereby creating a steep intracellular PI(3,4,5)P(3) gradient. Molecules involved in positive feedback signaling at the leading edge include the small G-proteins Rac and Ras, phosphatidylinositol-3 kinase and F-actin, as part of interlinked feedback loops that lead to a robust production of PI(3,4,5)P(3).

Actins↗
PubMed2006

Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR.

Directed cell migration and cell polarity are crucial in many facets of biological processes. Cellular motility requires a complex array of signaling pathways, in which orchestrated cross-talk, a feedback loop, and multi-component signaling recur. Almost every signaling molecule requires several regulatory processes to be functionally activated, and a lack of a signaling molecule often leads to chemotaxis defects, suggesting an integral role for each component in the pathway. We outline our current understanding of the signaling event that regulates chemotaxis with an emphasis on recent findings associated with the Ras, PI3K, and target of rapamycin (TOR) pathways and the interplay of these pathways. Ras, PI3K, and TOR are known as key regulators of cellular growth. Deregulation of those pathways is associated with many human diseases, such as cancer, developmental disorders, and immunological deficiency. Recent studies in yeast, mammalian cells, and Dictyostelium discoideum reveal another critical role of Ras, PI3K, and TOR in regulating the actin cytoskeleton, cell polarity, and cellular movement. These findings shed light on the mechanism by which eukaryotic cells maintain cell polarity and directed cell movement, and also demonstrate that multiple steps in the signal transduction pathway coordinately regulate cell motility.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Assaying chemotaxis of Dictyostelium cells.

Both prokaryote and eukaryote cells can sense and move up chemical concentration gradients (chemotax). As a means of finding food sources during vegetative growth, Dictyostelium discoideum naturally chemotaxes toward chemicals released by bacteria. As part of its developmental life cycle, D. discoideum chemotaxes towards cAMP. This chapter describes protocols for using Dictyostelium to understand the cell biology behind and the signaling events necessary for eukaryotic amoeboid chemotaxis. The chapter includes analyses of random cell motility, directed motility up chemical gradients, cellular responses to uniform chemoattractant exposure, and the utility of fluorescent probes for chemotaxis signaling events. Random cell motility in the absence of chemoattractant is analyzed to decipher the properties of self-organizing pseudopodia extension and retraction. Monitoring chemotaxis toward cAMP and folate allows the determination of signaling events required for sensing a chemical gradient and moving in a directed, persistent manner up the gradient. Uniform chemoattractant exposure is employed to elucidate the immediate intracellular responses to chemoattractant stimulation. Finally, analyzing cells expressing fluorescent fusion proteins is vital to elucidating the location of signaling events during chemotaxis.

Animals↗
PubMed2005

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium.

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.

Amino Acid Sequence↗
PubMed2005

A Dictyostelium homologue of WASP is required for polarized F-actin assembly during chemotaxis.

The actin cytoskeleton controls the overall structure of cells and is highly polarized in chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a lesser degree in the cell's posterior. Wiskott-Aldrich syndrome protein (WASP) has emerged as a central player in controlling actin polymerization. We have investigated WASP function and its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of WASP is required for the localization of WASP. We demonstrated that the B domain binds to PI(4,5)P2 and PI(3,4,5)P3 with similar affinities. The interaction between the B domain and PI(3,4,5)P3 plays an important role for the localization of WASP to the leading edge in chemotaxing cells. Our results suggest that the spatial and temporal control of WASP localization and activation is essential for the regulation of directional motility.

Actins↗
PubMed2005

Rho Rocks PTEN.

Explore the source record for details and available documents.

Animals↗
PubMed2005

Loss of SMEK, a novel, conserved protein, suppresses MEK1 null cell polarity, chemotaxis, and gene expression defects.

MEK/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1(-) (MEK1 null) and erk1(-) cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1(-) and erk1(-) chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene and protein (smkA and SMEK, respectively), whose loss partially suppresses the mek1(-) chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK, but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via a nuclear localization signal (NLS) at its C terminus. The importance of SMEK's nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1(-) and erk1(-) cells. The misexpression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner.

Amino Acid Sequence↗
PubMed2005

Finding the way: directional sensing and cell polarization through Ras signalling.

Chemotactic eukaryotic cells have the unique ability to sense a shallow extracellular chemoattractant gradient and translate it into a steep intracellular gradient. For example, phosphoinositide-3,4,5-trisphosphate (PIP3), the product of phosphatidylinositol-3-kinase (PI3K), is accumulated at the leading edge but not the back of a polarized chemotaxing cell. This is partially controlled by the reciprocal, preferential localization of PI3K and PTEN to the membrane at the front and back, respectively. However, upstream events that control the localized activation and localization of PI3K and PTEN remain unclear. Recent findings indicate that Ras is important for activation of the PI3K pathway and regulation of directed cell movement and cell polarity. Ras is activated at the leading edge, and this local activation occurs without asymmetric localization of PI3K and PTEN or the F-actin cytoskeleton. In contrast, P13K localization is driven by F-actin polymerization. Thus, Ras functions as an essential part of the cell's compass acting upstream of PI3K while reciprocal localization of PI3K and PTEN amplify the PIP3 gradient, rather than create it. These observations suggest a positive feedback loop to amplify an initial PIP3 gradient in which localized F-actin polymerization recruits cytosolic PI3K to the leading edge, where it is activated by Ras to locally produce PIP3 that induces F-actin polymerization.

Animals↗
PubMed2004

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)P(3), 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)P(3) 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 cell's 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.

Actins↗
PubMed2004

Dictyostelium PAKc is required for proper chemotaxis.

We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis. PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the Gbetagamma binding domain of Saccharomyces cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane. In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 s. pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient. PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in misregulated kinase activation and chemotaxis defects. We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia. A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway.

Actins↗
PubMed2004

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.

Actins↗
PubMed2004

Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.

Dictyostelium discoideum grow unicellularly, but develop as multicellular organisms. At two stages of development, their underlying symmetrical pattern of cellular organization becomes disrupted. During the formation of the multicellular aggregate, individual non-polarized cells re-organize their cytoskeletal structures to sequester specific intracellular signaling elements for activation by and directed movement within chemoattractant gradients. Subsequently, response to secreted morphogens directs undifferentiated populations to adopt different cell fates. Using a combination of cellular, biochemical and molecular approaches, workers have now begun to understand the mechanisms that permit Dictyostelium (and other chemotactic cells) to move directionally in shallow chemoattractant gradients and the transcriptional regulatory pathways that polarize cell-fate choice and initiate pattern formation.

Animals↗
PubMed2003

Cell migration: integrating signals from front to back.

Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes. This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them, and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.

Actin Cytoskeleton↗
PubMed2003

The novel ankyrin-repeat containing kinase ARCK-1 acts as a suppressor of the Spalten signaling pathway during Dictyostelium development.

Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasBGTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.

14-3-3 Proteins↗
PubMed2003

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.

Actins↗
PubMed2003

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 cell's 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]).

Cell Cycle Proteins↗