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Methods in Molecular Biology (Clifton,... 2022The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions,...
The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folic acid. Under starved conditions, they lose their sensitivity to pterins and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including ease of growth, genetic tractability, and the conservation of mammalian signaling pathways. In this chapter, we describe the use of the under-agarose chemotaxis assay to understand the signaling pathways controlling directional sensing and motility in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect conserved pathways involved in eukaryotic chemotaxis.
Topics: Amoeba; Animals; Chemotaxis; Cyclic AMP; Dictyostelium; Pterins; Sepharose
PubMed: 34542861
DOI: 10.1007/978-1-0716-1661-1_16 -
Sub-cellular Biochemistry 2022Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups...
Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.
Topics: Actin Cytoskeleton; Amoeba; Dictyostelium; Phosphatidylinositol 3-Kinases; Pinocytosis
PubMed: 35378702
DOI: 10.1007/978-3-030-94004-1_3 -
Philosophical Transactions of the Royal... Feb 2019Macropinocytosis-the large-scale, non-specific uptake of fluid by cells-is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular... (Review)
Review
Macropinocytosis-the large-scale, non-specific uptake of fluid by cells-is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular ruffles around regions of membrane defined by a patch of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the activated forms of the small G-proteins Ras and Rac. When this ruffle closes, a vesicle of the medium is delivered to the cell interior for further processing. It is accepted that PIP3 is required for efficient macropinocytosis. Here, we assess the roles of Ras and Rac in Dictyostelium macropinocytosis. Gain-of-function experiments show that macropinocytosis is stimulated by persistent Ras activation and genetic analysis suggests that RasG and RasS are the key Ras proteins involved. Among the activating guanine exchange factors (GEFs), GefF is implicated in macropinocytosis by an insertional mutant. The individual roles of Rho family proteins are little understood but activation of at least some may be independent of PIP3. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
Topics: Dictyostelium; Monomeric GTP-Binding Proteins; Pinocytosis
PubMed: 30967009
DOI: 10.1098/rstb.2018.0150 -
International Review of Cell and... 2016Small GTPases of the Rho family are ubiquitous molecular switches involved in the regulation of most actin cytoskeleton dependent processes and many other processes not... (Review)
Review
Small GTPases of the Rho family are ubiquitous molecular switches involved in the regulation of most actin cytoskeleton dependent processes and many other processes not directly linked to actin. D. discoideum is a well-established model organism for studies of the actin cytoskeleton and its regulation by signal transduction pathways. D. discoideum is equipped with a complex repertoire of Rho signaling components, with 20 Rho GTPases, more than 100 regulators (including exchange factors, GTPase activating proteins and guanine nucleotide dissociation inhibitors), and nearly 80 effectors or components of effector complexes. In this review we examine the knowledge accumulated to date about proteins involved in Rho-regulated signaling pathways in D. discoideum, with an emphasis on functional studies. We integrate the information about individual components into defined signaling pathways, with a focus on three extensively investigated processes: chemotaxis, vesicle trafficking, and cytokinesis.
Topics: Dictyostelium; Protozoan Proteins; Signal Transduction; rho GTP-Binding Proteins
PubMed: 26940518
DOI: 10.1016/bs.ircmb.2015.10.004 -
The International Journal of... 2019Autophagy is subdivided into chaperone-mediated autophagy, microautophagy and macroautophagy and is a highly conserved intracellular degradative pathway. It is crucial... (Review)
Review
Autophagy is subdivided into chaperone-mediated autophagy, microautophagy and macroautophagy and is a highly conserved intracellular degradative pathway. It is crucial for cellular homeostasis and also serves as a response to different stresses. Here we focus on macroautophagy, which targets damaged organelles and large protein assemblies, as well as pathogenic intracellular microbes for destruction. During this process, cytosolic material becomes enclosed in newly generated double-membrane vesicles, the so-called autophagosomes. Upon maturation, the autophagosome fuses with the lysosome for degradation of the cargo. The basic molecular machinery that controls macroautophagy works in a sequential order and consists of the ATG1 complex, the PtdIns3K complex, the membrane delivery system, two ubiquitin-like conjugation systems, and autophagy adaptors and receptors. Since the different stages of macroautophagy from initiation to final degradation of cargo are tightly regulated and highly conserved across eukaryotes, simple model organisms in combination with a wide range of techniques contributed significantly to advance our understanding of this complex dynamic process. Here, we present the social amoeba Dictyostelium discoideum as an advantageous and relevant experimental model system for the analysis of macroautophagy.
Topics: Animals; Autophagosomes; Autophagy; Caenorhabditis elegans; Class II Phosphatidylinositol 3-Kinases; Cytosol; Dictyostelium; Drosophila melanogaster; Homeostasis; Lysosomes; Phagocytosis; Saccharomyces cerevisiae; Ubiquitin
PubMed: 31840786
DOI: 10.1387/ijdb.190186LE -
Nature Communications Feb 2023Self-organization of cells is central to a variety of biological systems and physical concepts of condensed matter have proven instrumental in deciphering some of their...
Self-organization of cells is central to a variety of biological systems and physical concepts of condensed matter have proven instrumental in deciphering some of their properties. Here we show that microphase separation, long studied in polymeric materials and other inert systems, has a natural counterpart in living cells. When placed below a millimetric film of liquid nutritive medium, a quasi two-dimensional, high-density population of Dictyostelium discoideum cells spontaneously assembles into compact domains. Their typical size of 100 μm is governed by a balance between competing interactions: an adhesion acting as a short-range attraction and promoting aggregation, and an effective long-range repulsion stemming from aerotaxis in near anoxic condition. Experimental data, a simple model and cell-based simulations all support this scenario. Our findings establish a generic mechanism for self-organization of living cells and highlight oxygen regulation as an emergent organizing principle for biological matter.
Topics: Dictyostelium; Chemotaxis
PubMed: 36781863
DOI: 10.1038/s41467-023-36395-2 -
International Journal of Molecular... Feb 2020is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After... (Review)
Review
is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, is in a position to become a leader in the field of calmodulin research.
Topics: Binding Sites; Calcium; Calcium Signaling; Calmodulin; Calmodulin-Binding Proteins; Dictyostelium; EF Hand Motifs; Humans; Models, Molecular; Protein Binding; Protozoan Infections; Protozoan Proteins
PubMed: 32054133
DOI: 10.3390/ijms21041210 -
The International Journal of... 2019The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis,... (Review)
Review
The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis, phagocytosis and macropinocytosis, intercellular adhesion, pattern formation, caspase-independent cell death and more recently autophagy and social evolution. Being a soil amoeba and professional phagocyte, thus exposed to a variety of potential pathogens, D. discoideum has also proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections. The finding that the Dictyostelium genome harbours several homologs of human genes responsible for a variety of diseases has stimulated their analysis, providing new insights into the mechanism of action of the encoded proteins and in some cases into the defect underlying the disease. Recent technological developments have covered the genetic gap between mammals and non-mammalian model organisms, challenging the modelling role of the latter. Is there a future for Dictyostelium discoideum as a model organism?
Topics: Animals; Cell Biology; Cell Movement; Chemotaxis; Dictyostelium; Genome; History, 20th Century; History, 21st Century; Humans; Models, Biological; Phagocytosis
PubMed: 31840772
DOI: 10.1387/ijdb.190128sb -
The International Journal of... 2019By protein quality control and degradation, the ubiquitin system drives many essential regulatory processes such as cell cycle and division, signalling, DNA replication... (Review)
Review
By protein quality control and degradation, the ubiquitin system drives many essential regulatory processes such as cell cycle and division, signalling, DNA replication and repair. Therefore, dysfunctions in the ubiquitin system lead to many human disease states. However, despite the immense progress made over the last couple of decades, it appears that the ubiquitin system is more complex and multi-faced than formerly expected. In addition to a rich repertoire of ubiquitin, ubiquitin conjugating and de-ubiquitylating enzymes, the social amoeba Dictyostelium discoideum genome encodes also for a wide array of ubiquitin binding domain-containing proteins, thus offering the possibility to explore the biology of the ubiquitin system from cell and molecular biology points of view. We here provide an overview on the current knowledge about the Ub-system components and we discuss how Dictyostelium might be an outstanding eukaryotic cell model for unravelling the still mostly unknown ubiquitination mechanisms of some human diseases.
Topics: Animals; Cell Biology; Dictyostelium; Humans; Mice; Models, Biological; Plasmids; Proteasome Endopeptidase Complex; Protein Binding; Protein Domains; Protein Processing, Post-Translational; Proteolysis; RNA Interference; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31840790
DOI: 10.1387/ijdb.190260eb -
The International Journal of... 2019The social amoeba Dictyostelium discoideum is a tractable model organism to study cellular allorecognition, which is the ability of a cell to distinguish itself and its... (Review)
Review
The social amoeba Dictyostelium discoideum is a tractable model organism to study cellular allorecognition, which is the ability of a cell to distinguish itself and its genetically similar relatives from more distantly related organisms. Cellular allorecognition is ubiquitous across the tree of life and affects many biological processes. Depending on the biological context, these versatile systems operate both within and between individual organisms, and both promote and constrain functional heterogeneity. Some of the most notable allorecognition systems mediate neural self-avoidance in flies and adaptive immunity in vertebrates. D. discoideum's allorecognition system shares several structures and functions with other allorecognition systems. Structurally, its key regulators reside at a single genomic locus that encodes two highly polymorphic proteins, a transmembrane ligand called TgrC1 and its receptor TgrB1. These proteins exhibit isoform-specific, heterophilic binding across cells. Functionally, this interaction determines the extent to which co-developing D. discoideum strains co-aggregate or segregate during the aggregation phase of multicellular development. The allorecognition system thus affects both development and social evolution, as available evidence suggests that the threat of developmental cheating represents a primary selective force acting on it. Other significant characteristics that may inform the study of allorecognition in general include that D. discoideum's allorecognition system is a continuous and inclusive trait, it is pleiotropic, and it is temporally regulated.
Topics: Adaptive Immunity; Cell Adhesion; Chemotaxis; Dictyostelium; Glycoproteins; Glycosylation; Ligands; Models, Biological; Phenotype; Protozoan Proteins
PubMed: 31840777
DOI: 10.1387/ijdb.190239gs