Leep2A and Leep2B function as a RasGAP complex to regulate macropinosome formation.

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.

Self-organization of PIP3 waves is controlled by the topology and curvature of cell membranes.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results.

Deletion of Dictyostelium tpc2 gene forms multi-tipped structures, regulates autophagy and cell-type patterning.

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.

Cellular inertia.

It has been experimentally reported that chemotactic cells exhibit cellular memory, that is, a tendency to maintain the migration direction despite changes in the chemoattractant gradient. In this study, we analyzed a phenomenological model assuming the presence of cellular inertia, as well as a response time in motility, resulting in the reproduction of the cellular memory observed in the previous experiments. According to the analysis, the cellular motion is described by the superposition of multiple oscillative functions induced by the multiplication of the oscillative polarity and motility. The cellular intertia generates cellular memory by regulating phase differences between those oscillative functions. By applying the theory to the experimental data, the cellular inertia was estimated at [Formula: see text] min. In addition, physiological parameters, such as response time in motility and intracellular processing speed, were also evaluated. The agreement between the experiemental data and theory suggests the possibility of the presence of the response time in motility, which has never been biologically verified and should be explored in the future.

Decanoic Acid Stimulates Autophagy in D. discoideum.

Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in Dictyostelium, with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim (valproic acid), also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space.

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.

Identifying the Effects of Reactive Oxygen Species on Mitochondrial Dynamics and Cytoskeleton Stability in Dictyostelium discoideum.

Defects in mitochondrial dynamics, fission, fusion, and motility have been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and Charcot-Marie-Tooth disease. Another key feature of neurodegeneration is the increase in reactive oxygen species (ROS). Previous work has shown that the cytoskeleton, in particular the microtubules, and ROS generated by rotenone significantly regulate mitochondrial dynamics in Dictyostelium discoideum. The goal of this project is to study the effects of ROS on mitochondrial dynamics within our model organism D. discoideum to further understand the underlying issues that are the root of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We chose three likely ROS inducers, cumene hydroperoxide, hydroxylamine hydrochloride, and Antimycin A. Our work demonstrates that alteration of the microtubule cytoskeleton is not required to alter dynamics in response to ROS and there is no easy way to predict how mitochondrial dynamics will be altered based on which ROS generator is used. This research contributes to the better understanding of the cellular mechanisms that induce the pathogenesis of incurable neurodegenerative diseases with the hope that it will translate into developing new and more effective treatments for patients afflicted by them.

Phosphatidylserine exposure promotes increased adhesion in Dictyostelium Copine A mutants.

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.

Role of epigenetics in unicellular to multicellular transition in Dictyostelium.

BACKGROUND: The evolution of multicellularity is a critical event that remains incompletely understood. We use the social amoeba, Dictyostelium discoideum, one of the rare organisms that readily transits back and forth between both unicellular and multicellular stages, to examine the role of epigenetics in regulating multicellularity. RESULTS: While transitioning to multicellular states, patterns of H3K4 methylation and H3K27 acetylation significantly change. By combining transcriptomics, epigenomics, chromatin accessibility, and orthologous gene analyses with other unicellular and multicellular organisms, we identify 52 conserved genes, which are specifically accessible and expressed during multicellular states. We validated that four of these genes, including the H3K27 deacetylase hdaD, are necessary and that an SMC-like gene, smcl1, is sufficient for multicellularity in Dictyostelium. CONCLUSIONS: These results highlight the importance of epigenetics in reorganizing chromatin architecture to facilitate multicellularity in Dictyostelium discoideum and raise exciting possibilities about the role of epigenetics in the evolution of multicellularity more broadly.

Reverse fountain flow of phosphatidylinositol-3,4-bisphosphate polarizes migrating cells.

The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.

Sulfur sequestration promotes multicellularity during nutrient limitation.

The behaviour of Dictyostelium discoideum depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This biology makes D. discoideum an ideal model for investigating how fundamental metabolism commands cell differentiation and function. Here we show that reactive oxygen species-generated as a consequence of nutrient limitation-lead to the sequestration of cysteine in the antioxidant glutathione. This sequestration limits the use of the sulfur atom of cysteine in processes that contribute to mitochondrial metabolism and cellular proliferation, such as protein translation and the activity of enzymes that contain an iron-sulfur cluster. The regulated sequestration of sulfur maintains D. discoideum in a nonproliferating state that paves the way for multicellular development. This mechanism of signalling through reactive oxygen species highlights oxygen and sulfur as simple signalling molecules that dictate cell fate in an early eukaryote, with implications for responses to nutrient fluctuations in multicellular eukaryotes.

Cellular memory in eukaryotic chemotaxis depends on the background chemoattractant concentration.

Cells of the social amoeba Dictyostelium discoideum migrate to a source of periodic traveling waves of chemoattractant as part of a self-organized aggregation process. An important part of this process is cellular memory, which enables cells to respond to the front of the wave and ignore the downward gradient in the back of the wave. During this aggregation, the background concentration of the chemoattractant gradually rises. In our microfluidic experiments, we exogenously applied periodic waves of chemoattractant with various background levels. We find that increasing background does not make detection of the wave more difficult, as would be naively expected. Instead, we see that the chemotactic efficiency significantly increases for intermediate values of the background concentration but decreases to almost zero for large values in a switch-like manner. These results are consistent with a computational model that contains a bistable memory module, along with a nonadaptive component. Within this model, an intermediate background level helps preserve directed migration by keeping the memory activated, but when the background level is higher, the directional stimulus from the wave is no longer sufficient to activate the bistable memory, suppressing directed migration. These results suggest that raising levels of chemoattractant background may facilitate the self-organized aggregation in Dictyostelium colonies.

Cell dispersal by localized degradation of a chemoattractant.

Chemotaxis, the guided motion of cells by chemical gradients, plays a crucial role in many biological processes. In the social amoeba Dictyostelium discoideum, chemotaxis is critical for the formation of cell aggregates during starvation. The cells in these aggregates generate a pulse of the chemoattractant, cyclic adenosine 3',5'-monophosphate (cAMP), every 6 min to 10 min, resulting in surrounding cells moving toward the aggregate. In addition to periodic pulses of cAMP, the cells also secrete phosphodiesterase (PDE), which degrades cAMP and prevents the accumulation of the chemoattractant. Here we show that small aggregates of Dictyostelium can disperse, with cells moving away from instead of toward the aggregate. This surprising behavior often exhibited oscillatory cycles of motion toward and away from the aggregate. Furthermore, the onset of outward cell motion was associated with a doubling of the cAMP signaling period. Computational modeling suggests that this dispersal arises from a competition between secreted cAMP and PDE, creating a cAMP gradient that is directed away from the aggregate, resulting in outward cell motion. The model was able to predict the effect of PDE inhibition as well as global addition of exogenous PDE, and these predictions were subsequently verified in experiments. These results suggest that localized degradation of a chemoattractant is a mechanism for morphogenesis.

A Proteomics Analysis of Calmodulin-Binding Proteins in Dictyostelium discoideum during the Transition from Unicellular Growth to Multicellular Development.

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 Dictyostelium discoideum. Our analysis revealed several known CaMBPs in Dictyostelium 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 Dictyostelium 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 Dictyostelium, 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 Dictyostelium and sheds additional light on the essential roles of CaM and CaMBPs in eukaryotes.

Aggregative cycles evolve as a solution to conflicts in social investment.

Multicellular organization is particularly vulnerable to conflicts between different cell types when the body forms from initially isolated cells, as in aggregative multicellular microbes. Like other functions of the multicellular phase, coordinated collective movement can be undermined by conflicts between cells that spend energy in fuelling motion and 'cheaters' that get carried along. The evolutionary stability of collective behaviours against such conflicts is typically addressed in populations that undergo extrinsically imposed phases of aggregation and dispersal. Here, via a shift in perspective, we propose that aggregative multicellular cycles may have emerged as a way to temporally compartmentalize social conflicts. Through an eco-evolutionary mathematical model that accounts for individual and collective strategies of resource acquisition, we address regimes where different motility types coexist. Particularly interesting is the oscillatory regime that, similarly to life cycles of aggregative multicellular organisms, alternates on the timescale of several cell generations phases of prevalent solitary living and starvation-triggered aggregation. Crucially, such self-organized oscillations emerge as a result of evolution of cell traits associated to conflict escalation within multicellular aggregates.

Abundantly expressed class of noncoding RNAs conserved through the multicellular evolution of dictyostelid social amoebas.

Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia.

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells.

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosisD. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

Cytotoxicity and Mitochondrial Dysregulation Caused by α-Synuclein in Dictyostelium discoideum.

Alpha synuclein has been linked to both sporadic and familial forms of Parkinson's disease (PD) and is the most abundant protein in Lewy bodies a hallmark of Parkinson's disease. The function of this protein and the molecular mechanisms underlying its toxicity are still unclear, but many studies have suggested that the mechanism of α-synuclein toxicity involves alterations to mitochondrial function. Here we expressed human α-synuclein and two PD-causing α-synuclein mutant proteins (with a point mutation, A53T, and a C-terminal 20 amino acid truncation) in the eukaryotic model Dictyostelium discoideum. Mitochondrial disease has been well studied in D. discoideum and, unlike in mammals, mitochondrial dysfunction results in a clear set of defective phenotypes. These defective phenotypes are caused by the chronic hyperactivation of the cellular energy sensor, AMP-activated protein kinase (AMPK). Expression of α-synuclein wild type and mutant forms was toxic to the cells and mitochondrial function was dysregulated. Some but not all of the defective phenotypes could be rescued by down regulation of AMPK revealing both AMPK-dependent and -independent mechanisms. Importantly, we also show that the C-terminus of α-synuclein is required and sufficient for the localisation of the protein to the cell cortex in D. discoideum.

Superoxide dismutase C affects Dictyostelium contractile vacuole biogenesis and function.

Dictyostelium cells cope with hypo-osmotic stress with a contractile vacuole (CV) system, which consists of one or two vacuoles that cyclically charge and discharge. Uniquely, a F-Actin remodeling dependent minimal mixing of the CV membrane components with the target plasmalemma during the fusion and the dischargement warrants the integrity of the CV bladder for an efficient next CV cycle. The effect of hypo-osmotic stress on F-Actin remodeling activity, however, is currently not well understood. Dictyostelium cells increase the level of intracellular superoxide level in response to hypo-osmotic stress, which in turn activates redox-sensitive Ras proteins, but not Akt, which is one of the Ras downstream targets and a major regulator of F-Actin remodeling. However, Akt is not insulated from the active Ras in cells lacking Superoxide dismutase C (SodC). We report here that sodC- cells were compromised in the CV structure and function and the attenuation of Ras/PI3K/Akt signaling in several independent means significantly improved the compromised CV structure but not the function. Interestingly, when sodC- cells were treated with 5-(N,N-Dimethyl) amiloride hydrochloride (EIPA), an inhibitor of sodium proton exchanger (NHE), both the structure and the function of the CV improved. Thus, a proper CV biogenesis in sodC- cells was insufficient to restore their CV function, which in turn indicates the presence of an additional target for SodC and EIPA that modulates CV function.