Cell behaviors within a confined adhesive area fabricated using novel micropatterning methods.

In the field of cell and tissue engineering, there is an increasing demand for techniques to spatially control the adhesion of cells to substrates of desired sizes and shapes. Here, we describe two novel methods for fabricating a substrate for adhesion of cells to a defined area. In the first method, the surface of the coverslip or plastic dish was coated with Lipidure, a non-adhesive coating material, and air plasma was applied through a mask with holes, to confer adhesiveness to the surface. In the second method, after the surface of the coverslip was coated with gold by sputtering and then with Lipidure; the Lipidure coat was locally removed using a novel scanning laser ablation method. These methods efficiently confined cells within the adhesive area and enabled us to follow individual cells for a longer duration, compared to the currently available commercial substrates. By following single cells within the confined area, we were able to observe several new aspects of cell behavior in terms of cell division, cell-cell collisions, and cell collision with the boundary between adhesive and non-adhesive areas.

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent.

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein-coupled receptor GrlH, the G protein subunits Gß and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal-regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.

Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation.

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit-but not Scar-is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation-cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

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.

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.

GPCR Signaling Regulation in Dictyostelium Chemotaxis.

GPCR signaling is the most prevailing molecular mechanism for detecting ambient signals in eukaryotes. Chemotactic cells use GPCR signaling to process chemical cues for directional migration over a broad concentration range and with high sensitivity. Dictyostelium discoideum is a classical model, in which the molecular mechanism underlying eukaryotic chemotaxis has been well studied. Here, we describe protocols to evaluate the spatiotemporal chemotactic responses of Dictyostelium discoideum by different microscopic observations combined with biochemical assays. First, two different chemotaxis assays are presented to measure the dynamic concentration ranges for different cell strains or chemotactic parameters. Next, live-cell imaging and biochemical assays are provided to detect the activities of GPCR and its partner heterotrimeric G proteins upon chemoattractant stimulation. Finally, a method for detecting how a cell deciphers chemical gradients is described.

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.

Inhibiting Neddylation with MLN4924 Suppresses Growth and Delays Multicellular Development in Dictyostelium discoideum.

Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.

A sub-population of Dictyostelium discoideum cells shows extremely high sensitivity to cAMP for directional migration.

The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 µm wide, 5 µm high, and 100 µm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500-1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 µM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.

Methylation-directed acetylation of histone H3 regulates developmental sensitivity to histone deacetylase inhibition.

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug.

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.

Signaling interplay between PARP1 and ROS regulates stress-induced cell death and developmental changes in Dictyostelium discoideum.

Poly (ADP-ribose) polymerase-1 (PARP1) is a DNA damage sensor that gets activated in proportion to the damage, helping cells to determine whether to repair the damage or initiate cell death processes. We have previously shown PARP1's significance in the developmental processes of Dictyostelium discoideum in addition to its role in oxidative stress and UV-C stress induced cell death. In this study, we show the significance of ROS in PARP1 mediated responses of D. discoideum under different stress conditions. Interestingly, our results suggest differential kinetics of PARP1 activation and implications of ROS in starvation and cadmium induced cell death events. Increased accumulation of Poly (ADP-ribose), a product of PARP activation, could be detected within minutes post cadmium stress, whereas PARP1 activation was only a later event with starvation. Starvation induced PARP1 activation was supported by the depletion of ATP and NAD+, while PARP inhibitor confers protective effect during starvation. During starvation, cell death is induced in two phases, a primary ROS driven PARP1 independent early necrotic phase followed by a PARP1 driven ROS dependent paraptotic phase; both of which comprise mitochondrial changes. Cadmium (Cd) exerted a dose-dependent effect on cell death; a low dose of 0.2 mM Cd led to paraptosis and a higher dose of 0.5 mM Cd led to necrosis in D. discoideum cells within 24 h. Interestingly, glutathione (GSH) exposure could rescue cells from Cd stress mediated cell death. Besides unicellular cell death, the developmental arrest induced by cadmium and oxidative stress could be rescued by reinstating the redox equilibrium using GSH. In conclusion, we underscore the significant link between PARP1 and ROS in regulating the process of cell death and development in D. discoideum.

Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling.

Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.

Novel micropatterning technique reveals dependence of cell-substrate adhesion and migration of social amoebas on parental strain, development, and fluorescent markers.

Cell-substrate adhesion of the social amoeba Dictyostelium discoideum, a model organism often used for the study of chemotaxis, is non-specific and does not involve focal adhesion complexes. Therefore, micropatterned substrates where adherent Dictyostelium cells are constrained to designated microscopic regions are difficult to make. Here we present a micropatterning technique for Dictyostelium cells that relies on coating the substrate with an ∼1µm thick layer of polyethylene glycol (PEG) gel. We show that, when plated on a substrate with narrow parallel stripes of PEG-gel and glass, Dictyostelium cells nearly exclusive adhere to and migrate along the glass stripes, thus providing a model system to study one-dimensional migration of amoeboid cells. Surprisingly, we find substantial differences in the adhesion to PEG-gel and glass stripes between vegetative and developed cells and between two different axenic laboratory strains of Dictyostelium, AX2 and AX4. Even more surprisingly, we find that the distribution of Dictyostelium cells between PEG-gel and glass stripes is significantly affected by the expression of several fluorescent protein markers of the cytoskeleton. We carry out atomic force microscopy based single cell force spectroscopy measurements that confirm that the force of adhesion to PEG-gel substrate can be significantly different between vegetative and developed cells, AX2 and AX4 cells, and cells with and without fluorescent markers. Thus, the choice of parental background, the degree of development, and the expression of fluorescent protein markers can all have a profound effect on cell-substrate adhesion and should be considered when comparing migration of cells and when designing micropatterned substrates.

Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects.

Cancer cells display novel characteristics which can be exploited for therapeutic advantage. Isolated studies have shown that 1) the mevalonate pathway and 2) increased macropinocytosis are important in tumorigenesis, but a connection between these two observations has not been envisioned. A library screen for compounds that selectively killed Dictyostelium pten- cells identified pitavastatin. Pitavastatin also killed human breast epithelial MCF10A cells lacking PTEN or expressing K-RasG12V, as well as mouse tumor organoids. The selective killing of cells with oncogenic defects was traced to GGPP (geranylgeranyl diphosphate) depletion. Disruption of GGPP synthase in Dictyostelium revealed that GGPP is needed for pseudopod extension and macropinocytosis. Fluid-phase uptake through macropinocytosis is lower in PTEN-deleted cells and, as reported previously, higher in cells expressing activated Ras. Nevertheless, uptake was more sensitive to pitavastatin in cells with either of these oncogenic mutations than in wild-type cells. Loading the residual macropinosomes after pitavastatin with high concentrations of protein mitigated the cell death, indicating that defective macropinocytosis leads to amino acid starvation. Our studies suggest that the dependence of cancer cells on the mevalonate pathway is due to the role of GGPP in macropinocytosis and the reliance of these cells on macropinocytosis for nutrient uptake. Thus, inhibition of the networks mediating these processes is likely to be effective in cancer intervention.

Formins specify membrane patterns generated by propagating actin waves.

Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

THP-1 and Dictyostelium Infection Models for Screening and Characterization of Anti-Mycobacterium abscessus Hit Compounds.

!!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus 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 M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus 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 M. abscessus.

Two-photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment.

In this study, a two-photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil-living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two-photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.