Pharmacological inhibition of ENT1 enhances the impact of specific dietary fats on energy metabolism gene expression.

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.

Small molecule communication of Legionella: the ins and outs of autoinducer and nitric oxide signaling.

SUMMARYLegionella pneumophila is a Gram-negative environmental bacterium, which survives in planktonic form, colonizes biofilms, and infects protozoa. Upon inhalation of Legionella-contaminated aerosols, the opportunistic pathogen replicates within and destroys alveolar macrophages, thereby causing a severe pneumonia termed Legionnaires' disease. Gram-negative bacteria employ low molecular weight organic compounds as well as the inorganic gas nitric oxide (NO) for cell-cell communication. L. pneumophila produces, secretes, and detects the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one). LAI-1 is secreted by L. pneumophila in outer membrane vesicles and not only promotes communication among bacteria but also triggers responses from eukaryotic cells. L. pneumophila detects NO through three different receptors, and signaling through the volatile molecule translates into fluctuations of the intracellular second messenger cyclic-di-guanylate monophosphate. The LAI-1 and NO signaling pathways are linked via the pleiotropic transcription factor LvbR. In this review, we summarize current knowledge about inter-bacterial and inter-kingdom signaling through LAI-1 and NO by Legionella species.

A Mutant of Dictyostelium discoideum, KI-Cell, as a Model of Collective Cell Migration Independent of Chemotaxis.

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.

Electrotaxis of Dictyostelium discoideum, Migration in an Electric Field.

Living cells have the ability to detect electric fields and respond to them with directed migratory movements. Many proteomic approaches have been adopted in the past to identify the molecular mechanism behind this cellular phenomenon. However, how the cells sense the electric stimulus and transduce it into directed cell migration is still under discussion. Many eukaryotic cells react to applied electric stimulation, including Dictyostelium discoideum cells. We use them as model system for studying cell migration in electric fields, also known as electrotaxis. Here we report the protocols that we developed for our experiments. Our experimental outcomes helped us to characterize: (i) the memory that cells have in a varying electric field, which we defined as temporal electric persistence; and (ii) the accelerating motion of cells along their paths over the electric exposure time. We also report on the analysis of the role that conditioned medium factor (CMF), a protein secreted by cells when they begin to starve, plays in the mechanism of electric sensing. The results of this study can contribute to the understanding of the electrical sensing of cells and its transduction into directed cell migration.

Pseudopod Tracking and Statistics During Cell Movement in Buffer and Chemotaxis.

Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods. The trajectories of cell movement depend on the size, rhythm, and direction of long series of pseudopods. These pseudopod properties are regulated by internal factors such as memory of previous directions and by external factors such as gradients of chemoattractants or electric currents. Here a simple method is described that defines the X, Y time coordinates of a pseudopod at the start and the end of the extension phase. The connection between the start and end of an extending pseudopod defines a vector, which is the input of different levels of analysis that defines cell movement. The primary information of the vector is its spatial length (pseudopod size), temporal length (extension time), extension rate (size divided by time), and direction. The second layer of information describes the sequence of two (or more) pseudopods: the direction of the second pseudopod relative to the direction of the first pseudopod, the start of the second pseudopod relative to the extension phase of the first pseudopod (the second starts while the first is still extending or after the first has stopped), and the alternating right/left extension of pseudopods. The third layer of information is provided by specific and detailed statistical analysis of these data and addresses question such as: is pseudopod extension in buffer in random direction or has the system internal directional memory, and how do shallow external electrical or chemical gradients bias the intrinsic pseudopod extension. The method is described for Dictyostelium, but has been used successfully for fast-moving neutrophils, slow-moving stem cells, and the fungus B.d. chytrid.

Proteomic Analysis of Dictyostelium discoideum by Mass Spectrometry.

The large-scale proteomic analysis of Dictyostelium discoideum has contributed to our understanding of intracellular as well as secreted proteins in this versatile model eukaryote. Mass spectrometry-based proteomic analysis is a robust, sensitive, and rapid analytical method for identification and characterization of proteins extracted from tissues, cells, cell fractions, or pull-down assays. The availability of core facilities which make proteomics inexpensive and easy to do has facilitated a wide range of research projects. In this chapter, we present a simple standard methodology to extract proteins and prepare samples from D. discoideum for mass spectrometry and methods to analyze the identified proteins.

Complex third-party effects in the Dictyostelium-Paraburkholderia symbiosis: prey bacteria that are eaten, carried or left behind.

Symbiotic interactions may change depending on third parties like predators or prey. Third-party interactions with prey bacteria are central to the symbiosis between Dictyostelium discoideum social amoeba hosts and Paraburkholderia bacterial symbionts. Symbiosis with inedible Paraburkholderia allows host D. discoideum to carry prey bacteria through the dispersal stage where hosts aggregate and develop into fruiting bodies that disperse spores. Carrying prey bacteria benefits hosts when prey are scarce but harms hosts when prey bacteria are plentiful, possibly because hosts leave some prey bacteria behind while carrying. Thus, understanding benefits and costs in this symbiosis requires measuring how many prey bacteria are eaten, carried and left behind by infected hosts. We found that Paraburkholderia infection makes hosts leave behind both symbionts and prey bacteria. However, the number of prey bacteria left uneaten was too small to explain why infected hosts produced fewer spores than uninfected hosts. Turning to carried bacteria, we found that hosts carry prey bacteria more often after developing in prey-poor environments than in prey-rich ones. This suggests that carriage is actively modified to ensure hosts have prey in the harshest conditions. Our results show that multi-faceted interactions with third parties shape the evolution of symbioses in complex ways.

Assaying Lysosomal Enzyme Activity in Dictyostelium discoideum.

Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, ß-galactosidase, α-glucosidase, ß-glucosidase, ß-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).

Superresolution Expansion Microscopy in Dictyostelium Amoebae.

Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.

Discovery and Quantitative Dissection of Cytokinesis Mechanisms Using Dictyostelium discoideum.

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.

Quantitative Analysis of the Inactivation Process of Internalized Bacteria in Dictyostelium Cells.

The understanding of the inactivation process of ingested bacteria by phagocytes is a key focus in the field of host-pathogen interactions. Dictyostelium is a model organism that has been at the forefront of uncovering the mechanisms underlying this type of interaction. In this study, we describe an assay designed to measure the inactivation of Klebsiella aerogenes in the phagosomes of Dictyostelium discoideum.

Methods to Assess Autophagic Activity in Dictyostelium.

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.

Evaluating the Activity of Ras/Rap GTPases in Dictyostelium.

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.

Analysis of cGMP Signaling in Dictyostelium.

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.

Biogenesis of lysosome-related organelles complex-2 is an evolutionarily ancient proto-coatomer complex.

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.

Exploring host-pathogen interactions in the Dictyostelium discoideum-Mycobacterium marinum infection model of tuberculosis.

Mycobacterium tuberculosis is a pathogenic mycobacterium that causes tuberculosis. Tuberculosis is a significant global health concern that poses numerous clinical challenges, particularly in terms of finding effective treatments for patients. Throughout evolution, host immune cells have developed cell-autonomous defence strategies to restrain and eliminate mycobacteria. Concurrently, mycobacteria have evolved an array of virulence factors to counteract these host defences, resulting in a dynamic interaction between host and pathogen. Here, we review recent findings, including those arising from the use of the amoeba Dictyostelium discoideum as a model to investigate key mycobacterial infection pathways. D. discoideum serves as a scalable and genetically tractable model for human phagocytes, providing valuable insights into the intricate mechanisms of host-pathogen interactions. We also highlight certain similarities between M. tuberculosis and Mycobacterium marinum, and the use of M. marinum to more safely investigate mycobacteria in D. discoideum.

TED: Enhancing Translation Efficiency in Bacterial Expression Systems.

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.

Ion Signaling in Cell Motility and Development in Dictyostelium discoideum.

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.

Trafficking of adhesion and aggregation-modulating proteins during the early stages of Dictyostelium development.

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the µ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes.

Procedures to Measure Dictyostelium Phagocytosis and Macropinocytosis.

Uptaking particulate objects and bulk liquid by eucaryotic cells is critical for their growth, survival, and defense. Dictyostelium is a model organism spearheaded to uncover mechanisms behind various types of uptaking activities. Here, we describe assays measuring phagocytosis and macropinocytosis using Dictyostelium discoideum.