RESUMEN
Pluripotent stem cells can differentiate into distinct cell types but the intracellular pathways controlling cell fate choice are not well understood. The social amoeba Dictyostelium discoideum is a simplified system to study choice preference as proliferating amoebae enter a developmental cycle upon starvation and differentiate into two major cell types, stalk and spores, organised in a multicellular fruiting body. Factors such as acidic vesicle pH predispose amoebae to one fate. Here we show that the mechanistic target of rapamycin complex 1 (mTORC1) pathway has a role in cell fate bias in Dictyostelium. Inhibiting the mTORC1 pathway activity by disruption of Rheb (activator Ras homolog enriched in brain), or treatment with the mTORC1 inhibitor rapamycin prior to development, biases cells to a spore cell fate. Conversely activation of the pathway favours stalk cell differentiation. The Set1 histone methyltransferase, responsible for histone H3 lysine4 methylation, in Dictyostelium cells regulates transcription at the onset of development. Disruption of Set1 leads to high mTORC1 pathway activity and stalk cell predisposition. The ability of the mTORC1 pathway to regulate cell fate bias of cells undergoing differentiation offers a potential target to increase the efficiency of stem cell differentiation into a particular cell type.
Asunto(s)
Diferenciación Celular , Dictyostelium , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Dictyostelium/metabolismo , Dictyostelium/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Sirolimus/farmacología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genéticaRESUMEN
Medium chain fatty acids are commonly consumed as part of diets for endurance sports and as medical treatment in ketogenic diets where these diets regulate energy metabolism and increase adenosine levels. However, the role of the equilibrative nucleoside transporter 1 (ENT1), which is responsible for adenosine transport across membranes in this process, is not well understood. Here, we investigate ENT1 activity in controlling the effects of two dietary medium chain fatty acids (decanoic and octanoic acid), employing the tractable model system Dictyostelium. We show that genetic ablation of three ENT1 orthologues unexpectedly improves cell proliferation specifically following decanoic acid treatment. This effect is not caused by increased adenosine levels triggered by both fatty acids in the presence of ENT1 activity. Instead, we show that decanoic acid increases expression of energy-related genes relevant for fatty acid ß-oxidation, and that pharmacological inhibition of ENT1 activity leads to an enhanced effect of decanoic acid to increase expression of tricarboxylicacid cycle and oxidative phosphorylation components. Importantly, similar transcriptional changes have been shown in the rat hippocampus during ketogenic diet treatment. We validated these changes by showing enhanced mitochondria load and reduced lipid droplets. Thus, our data show that ENT1 regulates the medium chain fatty acid-induced increase in cellular adenosine levels and the decanoic acid-induced expression of important metabolic enzymes in energy provision, identifying a key role for ENT1 proteins in metabolic effects of medium chain fatty acids.
Asunto(s)
Metabolismo Energético , Tranportador Equilibrativo 1 de Nucleósido , Adenosina/metabolismo , Adenosina/farmacología , Caprilatos/farmacología , Proliferación Celular/efectos de los fármacos , Dictyostelium/metabolismo , Dictyostelium/genética , Dictyostelium/efectos de los fármacos , Dieta Cetogénica , Grasas de la Dieta/farmacología , Grasas de la Dieta/metabolismo , Metabolismo Energético/efectos de los fármacos , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacosRESUMEN
Collective cell migration occurs when the orientation of cell polarity is aligned with each other in a group of cells. Such collective polarization depends on a reciprocal process between cell intrinsic mechanisms such as cell-cell adhesion and extracellular guidance mechanism such as wound healing and chemotaxis. As part of its development life cycle, individual single cells of Dictyostelium discoideum exhibit chemotaxis toward cAMP, which is secreted from a certain population of cells. During the formation of multicellular body by chemotaxis-dependent cell aggregation, D. discoideum is also known to relay on multiple cell-cell adhesion mechanisms. In particular, tail-following behavior at the contact site, called contact following of locomotion (CFL), plays a pivotal role on the formation of the multicellular body. However, whether and how CFL alone can lead to a formation of collective behavior was not well understood. KI cell is a mutant of D. discoideum that lacks all chemotactic activity. Yet, it can exhibit the CFL activity and show nontrivial collective cell migration. This mutant provides an excellent model system to analyze the mechanism of the CFL and the macroscopic phenomena brought by the CFL. This chapter describes protocols for using KI cell to understand the biophysics and cell biology behind the collective cell migration induced by CFL.
Asunto(s)
Movimiento Celular , Quimiotaxis , Dictyostelium , Dictyostelium/genética , Dictyostelium/fisiología , Dictyostelium/citología , Quimiotaxis/genética , Movimiento Celular/genética , Mutación , AMP Cíclico/metabolismo , Polaridad Celular/genética , Adhesión Celular , Modelos BiológicosRESUMEN
Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought.
Asunto(s)
Dictyostelium , Lisosomas , Proteínas Protozoarias , Dictyostelium/genética , Dictyostelium/metabolismo , Lisosomas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Evolución Molecular , Proteína Coatómero/genética , Proteína Coatómero/metabolismo , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismoRESUMEN
Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate effects of Ras activity on cell motility or polarity, we focused on RasGAPs, C2GAPB in Dictyostelium amoebae and RASAL3 in HL-60 neutrophils and macrophages. In both cellular systems, optically recruiting the respective RasGAP to the cell front extinguished pre-existing protrusions and changed migration direction. However, when these respective RasGAPs were recruited uniformly to the membrane, cells polarized and moved more rapidly, whereas targeting to the back exaggerated these effects. These unexpected outcomes of attenuating Ras activity naturally had strong, context-dependent consequences for chemotaxis. The RasGAP-mediated polarization depended critically on myosin II activity and commenced with contraction at the cell rear, followed by sustained mTORC2-dependent actin polymerization at the front. These experimental results were captured by computational simulations in which Ras levels control front- and back-promoting feedback loops. The discovery that inhibiting Ras activity can produce counterintuitive effects on cell migration has important implications for future drug-design strategies targeting oncogenic Ras.
Asunto(s)
Actomiosina , Movimiento Celular , Polaridad Celular , Dictyostelium , Proteínas ras , Dictyostelium/metabolismo , Dictyostelium/genética , Células HL-60 , Actomiosina/metabolismo , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Macrófagos/metabolismo , Miosina Tipo II/metabolismo , Miosina Tipo II/genética , Neutrófilos/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Animales , Quimiotaxis , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Actinas/metabolismo , Simulación por Computador , Ratones , Transducción de SeñalRESUMEN
Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.
Asunto(s)
Membrana Celular , Dictyostelium , Lisosomas , Dictyostelium/genética , Dictyostelium/metabolismo , Membrana Celular/metabolismo , Lisosomas/metabolismo , Supervivencia CelularRESUMEN
The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.
Asunto(s)
Citocinesis , Dictyostelium , Dictyostelium/metabolismo , Dictyostelium/genética , Dictyostelium/citología , Biblioteca de Genes , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Recuperación de Fluorescencia tras Fotoblanqueo/métodosRESUMEN
Identifying the mechanisms of action of existing and novel drugs is essential for the development of new compounds for therapeutic and commercial use. Here we provide a technique to identify these mechanisms through isolating mutant cell lines that show resistance to drug-induced phenotypes using Dictyostelium discoideum REMI libraries. This approach provides a robust and rapid chemical-genetic screening technique that enables an unbiased approach to identify proteins and molecular pathways that control drug sensitivity. Mutations that result in drug resistance often occur in target proteins thus identifying the specific protein targets for drugs and bioactive natural products. Following the identification of a list of putative molecular targets user selected compound targets can be analyzed to confirm and validate direct inhibitory effects.
Asunto(s)
Dictyostelium , Mutación , Dictyostelium/genética , Dictyostelium/metabolismo , Enzimas de Restricción del ADN/metabolismo , Biblioteca de Genes , Resistencia a Medicamentos/genética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.
Asunto(s)
Dictyostelium , Proteínas ras , Dictyostelium/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Proteínas ras/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Unión ProteicaRESUMEN
Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.
Asunto(s)
GMP Cíclico , Dictyostelium , Transducción de Señal , Dictyostelium/metabolismo , Dictyostelium/genética , GMP Cíclico/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genéticaRESUMEN
Dictyostelium represents a stripped-down model for understanding how cells make decisions during development. The complete life cycle takes around a day and the fully differentiated structure is composed of only two major cell types. With this apparent reduction in "complexity," single cell transcriptomics has proven to be a valuable tool in defining the features of developmental transitions and cell fate separation events, even providing causal information on how mechanisms of gene expression can feed into cell decision-making. These scientific outputs have been strongly facilitated by the ease of non-disruptive single cell isolation-allowing access to more physiological measures of transcript levels. In addition, the limited number of cell states during development allows the use of more straightforward analysis tools for handling the ensuing large datasets, which provides enhanced confidence in inferences made from the data. In this chapter, we will outline the approaches we have used for handling Dictyostelium single cell transcriptomic data, illustrating how these approaches have contributed to our understanding of cell decision-making during development.
Asunto(s)
Dictyostelium , Perfilación de la Expresión Génica , Análisis de la Célula Individual , Transcriptoma , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Genetic engineering enables the forced expression of desired products in bacteria, which can then be used for a variety of applications, including functional analysis and pharmaceuticals. Here, we describe a method for tuning translation in bacteria, including Escherichia coli and Rhodobacter capsulatus, based on a phenomenon known as TED (translation enhancement by a Dictyostelium gene sequence). This method promotes translation of mRNA encoded by downstream genes by inserting a short nucleotide sequence into the 5' untranslated region between the promoter and the Shine-Dalgarno (SD) sequence. Various expression levels can be observed depending on the inserted sequence and its length, even with an identical promoter.
Asunto(s)
Escherichia coli , Biosíntesis de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones no Traducidas 5'/genética , Regiones Promotoras Genéticas , Dictyostelium/genética , Dictyostelium/metabolismo , Ingeniería Genética/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Autophagy is a degradative recycling process central to the maintenance of homeostasis in all eukaryotes. By ensuring the degradation of damaged mitochondria, it plays a key role in maintaining mitochondrial health and function. Of the highly conserved autophagy proteins, autophagy-related protein 1 (Atg1) is essential to the process. The involvement of these proteins in intracellular signalling pathways, including those involving mitochondrial function, are still being elucidated. Here the role of Atg1 was investigated in the simple model organism Dictyostelium discoideum using an atg1 null mutant and mutants overexpressing or antisense-inhibiting atg1. When evaluated against the well-characterised outcomes of mitochondrial dysfunction in this model, altered atg1 expression resulted in an unconventional set of phenotypic outcomes in growth, endocytosis, multicellular development, and mitochondrial homeostasis. The findings here show that Atg1 is involved in a tightly regulated signal transduction pathway coordinating energy-consuming processes such as cell growth and multicellular development, along with nutrient status and energy production. Furthermore, Atg1's effects on energy homeostasis indicate a peripheral ancillary role in the mitochondrial signalling network, with effects on energy balance rather than direct effects on electron transport chain function. Further research is required to tease out these complex networks. Nevertheless, this study adds further evidence to the theory that autophagy and mitochondrial signalling are not opposing but rather linked, yet strictly controlled, homeostatic mechanisms.
Asunto(s)
Autofagia , Dictyostelium , Endocitosis , Mitocondrias , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Dictyostelium/genética , Mitocondrias/metabolismo , Autofagia/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Homeostasis , Mutación/genéticaRESUMEN
Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.
Asunto(s)
Movimiento Celular , AMP Cíclico , Dictyostelium , Transducción de Señal , Dictyostelium/metabolismo , Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , Dictyostelium/citología , AMP Cíclico/metabolismo , Quimiotaxis , Comunicación Celular , Iones/metabolismo , Diferenciación Celular , Señalización del CalcioRESUMEN
Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.
Asunto(s)
Dictyostelium , Fosfohidrolasa PTEN , Fosfatos de Fosfatidilinositol , Proteínas Protozoarias , Dictyostelium/metabolismo , Dictyostelium/genética , Dictyostelium/enzimología , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Quimiotaxis , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins.
Asunto(s)
Adhesión Celular , Movimiento Celular , Quimiotaxis , Dictyostelium , Proteínas Protozoarias , Dictyostelium/genética , Dictyostelium/fisiología , Dictyostelium/metabolismo , Dictyostelium/crecimiento & desarrollo , Dictyostelium/citología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Sistemas CRISPR-Cas , Proteínas ras/metabolismo , Proteínas ras/genética , Edición Génica , Transducción de SeñalRESUMEN
Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni- to multicellular life. The D. firmibasis genome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-level D. firmibasis genome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison to Dictyostelium discoideum, the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.
Asunto(s)
Cromosomas , Dictyostelium , Genoma de Protozoos , Dictyostelium/genética , Anotación de Secuencia MolecularRESUMEN
Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation.
Asunto(s)
Altruismo , Dictyostelium , Proteínas de la Membrana , Proteínas Protozoarias , Quimiotaxis/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de Señal , Esporas Protozoarias/genética , Esporas Protozoarias/metabolismoRESUMEN
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
Asunto(s)
Dictyostelium , Diana Mecanicista del Complejo 2 de la Rapamicina , Transducción de Señal , Proteínas ras , Dictyostelium/metabolismo , Dictyostelium/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Proteínas ras/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , AMP Cíclico/metabolismoRESUMEN
BACKGROUND INFORMATION: Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. RESULTS: We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. CONCLUSION: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. SIGNIFICANCE: Decreased calcium due to deletion of tpc2 inhibit autophagic flux.