-
Disease Models & Mechanisms Jan 2018Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical...
Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.
Topics: Antioxidants; Curcumin; Dictyostelium; Ligands; Molecular Docking Simulation; Presenilin-1; Protein Phosphatase 2; Sequence Homology, Amino Acid
PubMed: 29361519
DOI: 10.1242/dmm.032375 -
Antimicrobial Agents and Chemotherapy Dec 2019presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. is able to grow intracellularly in human macrophages,...
presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for growth and a infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for .
Topics: Anti-Bacterial Agents; Dictyostelium; Humans; Macrophages; Microbial Sensitivity Tests; Mycobacterium abscessus; THP-1 Cells
PubMed: 31636068
DOI: 10.1128/AAC.01601-19 -
Environmental Science & Technology Sep 2022Amoebae are protists that are commonly found in water, soil, and other habitats around the world and have complex interactions with other microorganisms. In this work,...
Amoebae are protists that are commonly found in water, soil, and other habitats around the world and have complex interactions with other microorganisms. In this work, we investigated how host-endosymbiont interactions between amoebae and bacteria impacted the retention behavior of amoeba spores in porous media. A model amoeba species, , and a representative bacterium, B1qs70, were used to prepare amoeba spores that carried bacteria. After interacting with , the retention of spores was enhanced compared to noninfected spores. Diverse proteins, especially proteins contributing to the looser exosporium structure and cell adhesion functionality, are secreted in higher quantities on the exosporium surface of infected spores compared to that of noninfected ones. Comprehensive examinations using a quartz crystal microbalance with dissipation (QCM-D), a parallel plate chamber, and a single-cell force microscope present coherent evidence that changes in the exosporium of spores due to infection by enhance the connections between spores in the suspension and the spores that were previously deposited on the collector surface, thus resulting in more retention compared to the uninfected ones in porous media. This work provides novel insight into the retention of amoeba spores after bacterial infection in porous media and suggests that the host-endosymbiont relationship regulates the fate of biocolloids in drinking water systems, groundwater, and other porous environments.
Topics: Amoeba; Dictyostelium; Porosity; Spores, Bacterial; Symbiosis
PubMed: 35916900
DOI: 10.1021/acs.est.2c02899 -
Journal of Cell Science Apr 2022Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as...
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.
Topics: Animals; Dictyostelium; Endosomes; Humans; Mammals; Nutrients; Oligopeptides; Pinocytosis
PubMed: 35267018
DOI: 10.1242/jcs.259450 -
Cells Oct 2019Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify...
Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the unique advantages that this organism offers as a eukaryotic model for cell motility and as a nonmammalian model of human disease. By comparing whole-cell proteome analysis of developed (cAMP-pulsed) wild-type AX2 cells and an independent transcriptomic analysis of developed wild-type AX4 cells, our results show that up to 70% of the identified proteins overlap in the two independent studies. Among them, we have found 26 proteins previously related to cAMP signaling and identified 110 novel proteins involved in calcium signaling, adhesion, actin cytoskeleton, the ubiquitin-proteasome pathway, metabolism, and proteins that previously lacked any annotation. Our study validates previous findings, mostly for the canonical cAMP-pathway, and also generates further insight into the complexity of the transcriptomic changes during early development. This article also compares proteomic data between parental and cells lacking glkA, a GSK-3 kinase implicated in substrate adhesion and chemotaxis in Dictyostelium. This analysis reveals a set of proteins that show differences in expression in the two strains as well as overlapping protein level changes independent of GlkA.
Topics: Cell Differentiation; Dictyostelium; Gene Expression Profiling; Gene Expression Regulation, Developmental; Proteomics; Protozoan Proteins; Transcriptome
PubMed: 31581556
DOI: 10.3390/cells8101187 -
Frontiers in Cellular and Infection... 2017In recent years, has become an important model organism to study the cell biology of professional phagocytes. This amoeba not only shares many molecular features with... (Review)
Review
In recent years, has become an important model organism to study the cell biology of professional phagocytes. This amoeba not only shares many molecular features with mammalian macrophages, but most of its fundamental signal transduction pathways are conserved in humans. The broad range of existing genetic and biochemical tools, together with its suitability for cell culture and live microscopy, make an ideal and versatile laboratory organism. In this review, we focus on the use of as a phagocyte model for the study of mycobacterial infections, in particular . We look in detail at the intracellular cycle of , from its uptake by to its active or passive egress into the extracellular medium. In addition, we describe the molecular mechanisms that both the mycobacterial invader and the amoeboid host have developed to fight against each other, and compare and contrast with those developed by mammalian phagocytes. Finally, we introduce the methods and specific tools that have been used so far to monitor the - interaction.
Topics: Dictyostelium; Endocytosis; Host-Parasite Interactions; Microbiological Techniques; Mycobacterium marinum
PubMed: 29376033
DOI: 10.3389/fcimb.2017.00529 -
International Journal of Molecular... Feb 2021Calmodulin (CaM) is an essential calcium-binding protein within eukaryotes. CaM binds to calmodulin-binding proteins (CaMBPs) and influences a variety of cellular and...
Calmodulin (CaM) is an essential calcium-binding protein within eukaryotes. CaM binds to calmodulin-binding proteins (CaMBPs) and influences a variety of cellular and developmental processes. In this study, we used immunoprecipitation coupled with mass spectrometry (LC-MS/MS) to reveal over 500 putative CaM interactors in the model organism . Our analysis revealed several known CaMBPs in and mammalian cells (e.g., myosin, calcineurin), as well as many novel interactors (e.g., cathepsin D). Gene ontology (GO) term enrichment and Search Tool for the Retrieval of Interacting proteins (STRING) analyses linked the CaM interactors to several cellular and developmental processes in including cytokinesis, gene expression, endocytosis, and metabolism. The primary localizations of the CaM interactors include the nucleus, ribosomes, vesicles, mitochondria, cytoskeleton, and extracellular space. These findings are not only consistent with previous work on CaM and CaMBPs in , but they also provide new insight on their diverse cellular and developmental roles in this model organism. In total, this study provides the first in vivo catalogue of putative CaM interactors in and sheds additional light on the essential roles of CaM and CaMBPs in eukaryotes.
Topics: Calmodulin; Calmodulin-Binding Proteins; Cell Proliferation; Chromatography, High Pressure Liquid; Dictyostelium; Protein Interaction Mapping; Protein Interaction Maps; Proteomics; Protozoan Proteins; Tandem Mass Spectrometry
PubMed: 33572113
DOI: 10.3390/ijms22041722 -
Genome Biology and Evolution Feb 2018Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyosteliids, has evolved a relatively simple...
Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyosteliids, has evolved a relatively simple aggregative multicellular stage resulting in a fruiting body supported by a stalk. Protosteloid amoeba, which are scattered throughout the amoebozoan tree, differ by producing only one or few single stalked spores. Thus, one obvious difference in the developmental cycle of protosteliids and dictyosteliids seems to be the establishment of multicellularity. To separate spore development from multicellular interactions, we compared the genome and transcriptome of a Protostelium species (Protostelium aurantium var. fungivorum) with those of social and solitary members of the Amoebozoa. During fruiting body formation nearly 4,000 genes, corresponding to specific pathways required for differentiation processes, are upregulated. A comparison with genes involved in the development of dictyosteliids revealed conservation of >500 genes, but most of them are also present in Acanthamoeba castellanii for which fruiting bodies have not been documented. Moreover, expression regulation of those genes differs between P. aurantium and Dictyostelium discoideum. Within Amoebozoa differentiation to fruiting bodies is common, but our current genome analysis suggests that protosteliids and dictyosteliids used different routes to achieve this. Most remarkable is both the large repertoire and diversity between species in genes that mediate environmental sensing and signal processing. This likely reflects an immense adaptability of the single cell stage to varying environmental conditions. We surmise that this signaling repertoire provided sufficient building blocks to accommodate the relatively simple demands for cell-cell communication in the early multicellular forms.
Topics: Amoebozoa; Cell Communication; Dictyostelium; Evolution, Molecular; Gene Expression Regulation, Developmental; Phylogeny; Protozoan Proteins; Transcriptome
PubMed: 29378020
DOI: 10.1093/gbe/evy011 -
Journal of Molecular Evolution Feb 2023The study of spontaneous mutation rates has revealed a wide range of heritable point mutation rates across species, but there are comparatively few estimates for...
The study of spontaneous mutation rates has revealed a wide range of heritable point mutation rates across species, but there are comparatively few estimates for large-scale deletion and duplication rates. The handful of studies that have directly calculated spontaneous rates of deletion and duplication using mutation accumulation lines have estimated that genes are duplicated and deleted at orders of magnitude greater rates than the spontaneous point mutation rate. In our study, we tested whether spontaneous gene deletion and gene duplication rates are also high in Dictyostelium discoideum, a eukaryote with among the lowest point mutation rates (2.5 × 10 per site per generation) and an AT-rich genome (GC content of 22%). We calculated mutation rates of gene deletions and duplications using whole-genome sequencing data originating from a mutation accumulation experiment and determined the association between the copy number mutations and GC content. Overall, we estimated an average of 3.93 × 10 gene deletions and 1.18 × 10 gene duplications per gene per generation. While orders of magnitude greater than their point mutation rate, these rates are much lower compared to gene deletion and duplication rates estimated from mutation accumulation lines in other organisms (that are on the order of ~ 10 per gene/generation). The deletions and duplications were enriched in regions that were AT-rich even compared to the genomic background, in contrast to our expectations if low GC content was contributing to low mutation rates. The low deletion and duplication mutation rates in D. discoideum compared to other eukaryotes mirror their low point mutation rates, supporting previous work suggesting that this organism has high replication fidelity and effective molecular machinery to avoid the accumulation of mutations in their genome.
Topics: Gene Duplication; Dictyostelium; Gene Deletion; Mutation; Genome; Eukaryota
PubMed: 36484794
DOI: 10.1007/s00239-022-10081-1 -
ELife Oct 2019Eukaryotic cells can migrate using different modes, ranging from amoeboid-like, during which actin filled protrusions come and go, to keratocyte-like, characterized by a...
Eukaryotic cells can migrate using different modes, ranging from amoeboid-like, during which actin filled protrusions come and go, to keratocyte-like, characterized by a stable morphology and persistent motion. How cells can switch between these modes is not well understood but waves of signaling events are thought to play an important role in these transitions. Here we present a simple two-component biochemical reaction-diffusion model based on relaxation oscillators and couple this to a model for the mechanics of cell deformations. Different migration modes, including amoeboid-like and keratocyte-like, naturally emerge through transitions determined by interactions between biochemical traveling waves, cell mechanics and morphology. The model predictions are explicitly verified by systematically reducing the protrusive force of the actin network in experiments using cells. Our results indicate the importance of coupling signaling events to cell mechanics and morphology and may be applicable in a wide variety of cell motility systems.
Topics: Biochemical Phenomena; Biomechanical Phenomena; Cell Movement; Dictyostelium; Models, Biological
PubMed: 31625907
DOI: 10.7554/eLife.48478