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Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation., (© 2024 Chao et al.)

Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RasG is a major regulator of macropinocytosis in Dictyostelium discoideum . Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo , the physiological significance of the Rab5A-IqgC interaction remains elusive.

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells., (© 2023 Li et al.)

The Dictyostelium atypical mitogen-activated protein kinase (MAPK) Erk2 is required for chemotactic responses to cAMP as amoeba undergo multicellular development. In this study, Erk2 was found to be essential for the cAMP-stimulated translocation of the GATA transcription factor GtaC as indicated by the distribution of a GFP-GtaC reporter. Erk2 was also found to be essential for the translocation of GtaC in response to external folate, a foraging signal that directs the chemotaxis of amoeba to bacteria. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor Far1 or the coupled G-protein subunit Gα4. Loss of GtaC function resulted in enhanced chemotactic movement to folate, suggesting that GtaC suppresses responses to folate. The alteration of four Erk2-preferred phosphorylation sites in GtaC impacted the translocation of GFP-GtaC in response to folate and the GFP-GtaC-mediated rescue of aggregation and development of gtaC- cells. The ability of different chemoattractants to stimulate Erk2-regulated GtaC translocation suggests that atypical MAPK-mediated regulation of transcription factors can contribute to different cell fates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent macropinosomes mature after internalization remains largely unknown. By searching for proteins that localize on macropinosomes during the Rab5-to-Rab7 transition stage in Dictyostelium, we uncover a complex composed of two proteins, which we name PripA and TbcrA. We show that the Rab5-to-Rab7 conversion involves fusion of Rab5-marked early macropinosomes with Rab7-marked late macropinosomes. PripA links the two membrane compartments by interacting with PI(3,4)P 2 and Rab7. In addition, PripA recruits TbcrA, which acts as a GAP, to turn off Rab5. Thus, the conversion to Rab7 is linked to inactivation of the upstream Rab5. Consistently, disruption of either pripA or tbcrA impairs Rab5 inactivation and macropinocytic cargo processing. Therefore, the PripA-TbcrA complex is the central component of a Rab GAP cascade that facilitates programmed Rab switch and efficient cargo trafficking during macropinosome maturation., (© 2022. The Author(s).)

Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as a nutrient-scavenging pathway in mammalian cells. How environmental and cellular nutrient status modulates macropinocytic activity is not well understood. By developing a high-content imaging-based genetic screen in Dictyostelium discoideum we identified Slc15A, an oligopeptide transporter located at the plasma membrane and early macropinosome, as a novel macropinocytosis regulator. We show that deletion of slc15A but not two other related slc15 genes, leads to reduced macropinocytosis, reduced cell growth and aberrantly increased autophagy in cells grown in nutrient-rich medium. Expression of Slc15A protein or supplying cells with free amino acids rescues these defects. In contrast, expression of transport-defective Slc15A or supplying cells with amino acids in their di-peptide forms fails to rescue these defects. Therefore, Slc15A modulates the level of macropinocytosis by maintaining the intracellular availability of key amino acids through extraction of oligopeptides from the early macropinocytic pathway. We propose that Slc15A constitutes part of a positive feedback mechanism coupling cellular nutrient status and macropinocytosis. This article has an associated First Person interview with the first authors of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The diverse migratory modes displayed by different cell types are generally believed to be idiosyncratic. Here we show that the migratory behaviour of Dictyostelium was switched from amoeboid to keratocyte-like and oscillatory modes by synthetically decreasing phosphatidylinositol-4,5-bisphosphate levels or increasing Ras/Rap-related activities. The perturbations at these key nodes of an excitable signal transduction network initiated a causal chain of events: the threshold for network activation was lowered, the speed and range of propagating waves of signal transduction activity increased, actin-driven cellular protrusions expanded and, consequently, the cell migratory mode transitions ensued. Conversely, innately keratocyte-like and oscillatory cells were promptly converted to amoeboid by inhibition of Ras effectors with restoration of directed migration. We use computational analysis to explain how thresholds control cell migration and discuss the architecture of the signal transduction network that gives rise to excitability.

Directional sensing, a process in which cells convert an external chemical gradient into internal signaling events, is essential in chemotaxis. We previously showed that a Rho GTPase, RacE, regulates gradient sensing in Dictyostelium cells. Here, using affinity purification and mass spectrometry, we identify a novel RacE-binding protein, GflB, which contains a Ras GEF domain and a Rho GAP domain. Using biochemical and gene knockout approaches, we show that GflB balances the activation of Ras and Rho GTPases, which enables cells to precisely orient signaling events toward higher concentrations of chemoattractants. Furthermore, we find that GflB is located at the leading edge of migrating cells, and this localization is regulated by the actin cytoskeleton and phosphatidylserine. Our findings provide a new molecular mechanism that connects directional sensing and morphological polarization., (© 2016 Senoo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism.

Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Among these, we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8, or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis., (Copyright © 2015, American Association for the Advancement of Science.)

Biological oscillations are observed at many levels of cellular organization. In the social amoeba Dictyostelium discoideum, starvation-triggered multicellular development is organized by periodic cyclic adenosine 3',5'-monophosphate (cAMP) waves, which provide both chemoattractant gradients and developmental signals. We report that GtaC, a GATA transcription factor, exhibits rapid nucleocytoplasmic shuttling in response to cAMP waves. This behavior requires coordinated action of a nuclear localization signal and reversible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor-mediated phosphorylation. Although both are required for developmental gene expression, receptor occupancy promotes nuclear exit of GtaC, which leads to a transient burst of transcription at each cAMP cycle. We demonstrate that this biological circuit filters out high-frequency signals and counts those admitted, thereby enabling cells to modulate gene expression according to the dynamic pattern of the external stimuli.

Dictyostelium discoideum is an excellent model organism for the study of directed cell migration, since Dictyostelium cells show robust chemotactic responses to the chemoattractant cAMP. Many powerful experimental tools are applicable, including forward and reverse genetics, biochemistry, microscopy, and proteomics. Recent studies have demonstrated that many components involved in chemotaxis are functionally conserved between human neutrophils and Dictyostelium amoebae. In this chapter, we describe how to define the functions of proteins that mediate and regulate cell motility, cell polarity, and directional sensing during chemotaxis in Dictyostelium.

In chemotactic cells, G protein-coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasC(Q62L)-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2-PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

Abstract: Many cells have the ability to grow or migrate towards chemical cues. Oriented growth and movement require detection of the external chemical gradient, transduction of signals, and reorganization of the cytoskeleton. Recent studies in Dictyostelium discoideum and mammalian neutrophils have revealed a complex signaling network that enables cells to migrate in chemical gradients. [Copyright &y& Elsevier]