Dictyostelium Discoideum Cells Research Articles

Modeling cell crawling strategies with a bistable model: From amoeboid to fan-shaped cell motion

Eukaryotic cell motility involves a complex network of interactions between biochemical components and mechanical processes. The cell employs this network to polarize and induce shape changes that give rise to membrane protrusions and retractions, ultimately leading to locomotion of the entire cell body. The combination of a nonlinear reaction–diffusion model of cell polarization, noisy bistable kinetics, and a dynamic phase field for the cell shape permits us to capture the key features of this complex system to investigate several motility scenarios, including amoeboid and fan-shaped forms as well as intermediate states with distinct displacement mechanisms. We compare the numerical simulations of our model to live cell imaging experiments of motile Dictyostelium discoideum cells under different developmental conditions. The dominant parameters of the mathematical model that determine the different motility regimes are identified and discussed.

Evaluation of exosome loading characteristics in their purification via a glycerol-assisted hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber phase.

Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5-30 cm) and loading flow rates (0.2-0.5 ml/min). A loading capacity of 5.4 × 1012 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications.

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.

Shaping the Cytoskeleton with Electric Fields

Endogenous electric fields are present in many biological processes including at the edge of healing wounds and can direct cell migration. Here we measure the effect of DC electric fields on actin polymerization dynamics. We find that actin waves are guided by DC electric fields for multiple cell types. Giant Dictyostelium discoideum cells (produced via electrofusion) allow us to measure electric field response of actin waves located far from the leading or trailing edge of a cell, and thus decouple cell polarity and shape from actin wave responses. We also use nanoridges, fabricated substrate nanotopographies, which alter the actin waves from the broad, two-dimensional waves seen on flat substrates to one-dimensional waves traveling along the nanoridges. Our data reveal new insights into the mechanism behind cell migration via DC electric fields with actin waves playing a key role in cell response and control.

Unstable cAMP wave patterns during aggregation of Dictyostelium discoideum cells

Flow-driven formation of unstable patterns of cyclic adenosine monophosphate (cAMP) is investigated in the Martiel-Goldbeter (MG) model. This is predicted via a complex Ginzburg-Landau equation, derived from the MG model, under the so-called modulational instability process. Regions of parameters where patterns exist are discussed analytically and verified numerically. Quasi-periodic waves, spiral seeds and chaotic patterns are found to control information driven in a colony of homogeneously distributed Dictyostelium discoideum cells under the change of the extracellular cAMP degradation rate (ke), the production rate of cAMP (σ) and the advection flow velocity (Vf). Our results suggest that these quantities play a key role in the efficient regulation of communication within an amoeba colony, and the presence of the flow makes it possible to understand pattern formation process among D. discoideum cells under spontaneous fluid flow in their natural environment.

A flow-based microfluidic device for spatially quantifying intracellular calcium ion activity during cellular electrotaxis.

How a cell senses, responds, and moves toward, or away from an external cue is central to many biological and medical phenomena including morphogenesis, immune response, and cancer metastasis. Many eukaryotic cells have internal sensory mechanisms that allow them to sense these cues, often in the form of gradients of chemoattractant, voltage, or mechanical stress, and bias their motion in a specific direction. In this study, a new method for using microfluidics to study the electrotactic migration of cells is presented. Electrotaxis (also known as galvanotaxis) is the phenomenon by which cells bias their motion directionally in response to an externally applied electrical field. In this work, we present a new flow-based, salt bridge-free microfluidic device for imaging and quantifying cell motility and intracellular ion activity during electrotaxis. To eliminate salt bridges, we used a low nanoliter flow rate to slowly drive Faradaic waste products away from and out of the electrotaxis zone. This cell migration zone consisted of an array of fluidic confinement channels approximately 2 μm in thickness. This confined height served to insulate the migrating cells from the electric field at the top and bottom of the cell, such that only the two-dimensional perimeter of the cells interacted with the electrical source. We demonstrate the ability to quantify the electrotactic velocity of migrating Dictyostelium discoideum cells and show how this confined design facilitates the imaging and quantification of the ion activity of electrotaxing cells. Finally, by spatially imaging the calcium concentration within these cells, we demonstrate that intracellular calcium preferentially translocates to the leading edge of migrating Dictyostelium cells during electrotaxis but does not exhibit this behavior during migration by chemotaxis in a gradient of cyclic adenosine 3',5'-monophosphate or when cells freely migrate in the absence of an external cue.

Plasticity of cell migration resulting from mechanochemical coupling.

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 Dictyostelium discoideum 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.

Phosphorylated Rho-GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras-GTP to regulate cell migration.

mTORC2 plays critical roles in metabolism, cell survival, and actin cytoskeletal dynamics via phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae Dictyostelium cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling the supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by chemoattractant-induced phosphorylation of Rho-GDP at serine 192 by GSK-3. Furthermore, Rho-GDP rescued defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of serine 192. Thus, in contrast to the prevailing view that GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signaling is activated by Rho-GDP.

Heterogeneities Shape Passive Intracellular Transport

A living cell’s interior is one of the most complex and intrinsically dynamic systems, providing an elaborate interplay between cytosolic crowding and ATP-driven motion that controls cellular functionality. Here, we investigated two distinct fundamental features of the merely passive, non-biomotor-shuttled material transport within the cytoplasm of Dictyostelium discoideum cells: the anomalous non-linear scaling of the mean-squared displacement of a 150-nm-diameter particle and non-Gaussian distribution of increments. Relying on single-particle tracking data of 320,000 data points, we performed a systematic analysis of four possible origins for non-Gaussian transport: 1) sample-based variability, 2) rarely occurring strong motion events, 3) ergodicity breaking/aging, and 4) spatiotemporal heterogeneities of the intracellular medium. After excluding the first three reasons, we investigated the remaining hypothesis of a heterogeneous cytoplasm as cause for non-Gaussian transport. A, to our knowledge, novel fit model with randomly distributed diffusivities implementing medium heterogeneities suits the experimental data. Strikingly, the non-Gaussian feature is independent of the cytoskeleton condition and lag time. This reveals that efficiency and consistency of passive intracellular transport and the related anomalous scaling of the mean-squared displacement are regulated by cytoskeleton components, whereas cytoplasmic heterogeneities are responsible for the generic, non-Gaussian distribution of increments.

Force balances between interphase centrosomes as revealed by laser ablation.

Numerous studies have highlighted the self-centering activities of individual microtubule (MT) arrays in animal cells, but relatively few works address the behavior of multiple arrays that coexist in a common cytoplasm. In multinucleated Dictyostelium discoideum cells, each centrosome organizes a radial MT network, and these networks remain separate from one another. This feature offers an opportunity to reveal the mechanism(s) responsible for the positioning of multiple centrosomes. Using a laser microbeam to eliminate one of the two centrosomes in binucleate cells, we show that the unaltered array is rapidly repositioned at the cell center. This result demonstrates that each MT array is constantly subject to centering forces and infers a mechanism to balance the positions of multiple arrays. Our results address the limited actions of three kinesins and a cross-linking MAP that are known to have effects in maintaining MT organization and suggest a simple means used to keep the arrays separated.

Oscillatory cAMP cell-cell signalling persists during multicellular Dictyostelium development

Propagating waves of cAMP, periodically initiated in the aggregation centre, are known to guide the chemotactic aggregation of hundreds of thousands of starving individual Dictyostelium discoideum cells into multicellular aggregates. Propagating optical density waves, reflecting cell periodic movement, have previously been shown to exist in streaming aggregates, mounds and migrating slugs. Using a highly sensitive cAMP-FRET reporter, we have now been able to measure periodically propagating cAMP waves directly in these multicellular structures. In slugs cAMP waves are periodically initiated in the tip and propagate backward through the prespore zone. Altered cAMP signalling dynamics in mutants with developmental defects strongly support a key functional role for cAMP waves in multicellular Dictyostelium morphogenesis. These findings thus show that propagating cAMP not only control the initial aggregation process but continue to be the long range cell-cell communication mechanism guiding cell movement during multicellular Dictyostelium morphogenesis at the mound and slugs stages.

Effect Of Loading Method On Metabolism Of Foreign Molecules In Intact Cells Of Dictyostelium Discoideum

Loading foreign molecules inside living cells allows one to monitor cellular metabolism, track various metabolic pathways and alter activities of the cell. However, the effects of loading methods on the fate of these foreign molecules are poorly understood. In this experiment, we examined three promising loading methods including pinocytosis, electroporation and myristoylation to insert a peptide substrate reporter in cells of Dictyostelium discoideum. These methods were optimized to maximize cell viability, and factors such as loading efficiency, loading duration, localization and uniformity were characterized. Capillary electrophoresis was used to determine how each loading method affected the enzyme activity on the peptide substrate reporter. It was observed that these loading methods had strong effects on the stability and degradation of the peptide. Myristoylation was found to be the most effective method at transporting the peptide into cells while electroporation produced the most uniform loading within cells suggesting cytoplasmic loading. The peptide was degraded most rapidly after loading by myristoylation (half‐life = 12 ± 1 min), while pinocytic loading produced slightly slower degradation (half‐life = 19 ± 2 min). Phosphorylation of the peptide was only observed in cells loaded with electroporation. This experiment suggests that the choice of loading method influences the stability and metabolism of foreign molecules in cells. Our future work includes investigating the specificity of different peptide substrate reporters in Dictyostelium lysates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The putative G protein-coupled receptor GrlD mediates extracellular polyphosphate sensing in Dictyostelium discoideum.

Five or more orthophosphates bound together by high-energy phosphoanhydride bonds are highly ubiquitous inorganic molecules called polyphosphate. Polyphosphate acts as a signaling molecule eliciting a number of responses in eukaryotic cells, but the mechanisms mediating these effects are poorly understood. Proliferating Dictyostelium discoideum cells accumulate extracellular polyphosphate. At extracellular concentrations similar to those observed in stationary phase cells, polyphosphate inhibits proteasome activity and proliferation, and induces aggregation. Here we identify GrlD as a putative G protein–coupled receptor that mediates binding of extracellular polyphosphate to the cell surface. Cells lacking GrlD do not respond to polyphosphate-induced proteasome inhibition, aggregation, or proliferation inhibition. Polyphosphate also elicits differential effects on cell-substratum adhesion and cytoskeletal F-actin levels based on nutrient availability, and these effects were also mediated by GrlD. Starving cells also accumulate extracellular polyphosphate. Starved cells treated with exopolyphosphatase failed to aggregate effectively, suggesting that polyphosphate also acts as a signaling molecule during starvation-induced development of Dictyostelium. Together, these results suggest that a eukaryotic cell uses a G protein–coupled receptor to mediate the sensing and response to extracellular polyphosphate.

Harnessing Motile Amoeboid Cells as Trucks for Microtransport and -Assembly.

Cell‐driven microtransport is one of the most prominent applications in the emerging field of biohybrid systems. While bacterial cells have been successfully employed to drive the swimming motion of micrometer‐sized cargo particles, the transport capacities of motile adherent cells remain largely unexplored. Here, it is demonstrated that motile amoeboid cells can act as efficient and versatile trucks to transport microcargo. When incubated together with microparticles, cells of the social amoeba Dictyostelium discoideum readily pick up and move the cargo particles. Relying on the unspecific adhesive properties of the amoeba, a wide range of different cargo materials can be used. The cell‐driven transport can be directionally guided based on the chemotactic responses of amoeba to chemoattractant gradients. On the one hand, the cargo can be assembled into clusters in a self‐organized fashion, relying on the developmentally induced chemotactic aggregation of cells. On the other hand, chemoattractant gradients can be externally imposed to guide the cellular microtrucks to a desired location. Finally, larger cargo particles of different shapes that exceed the size of a single cell by more than an order of magnitude, can also be transported by the collective effort of large numbers of motile cells.

Ultraflexible Nanowire Array for Label- and Distortion-Free Cellular Force Tracking.

Living cells interact with their immediate environment by exerting mechanical forces, which regulate important cell functions. Elucidation of such force patterns yields deep insights into the physics of life. Here we present a top-down nanostructured, ultraflexible nanowire array biosensor capable of probing cell-induced forces. Its universal building block, an inverted conical semiconductor nanowire, greatly enhances both the functionality and the sensitivity of the device. In contrast to existing cellular force sensing architectures, microscopy is performed on the nanowire heads while cells deflecting the nanowires are confined within the array. This separation between the optical path and the cells under investigation excludes optical distortions caused by cell-induced refraction, which can give rise to feigned displacements on the 100 nm scale. The undistorted nanowire displacements are converted into cellular forces via the nanowire spring constant. The resulting distortion-free cellular force transducer realizes a high-resolution and label-free biosenor based on optical microscopy. Its performance is demonstrated in a proof-of-principle experiment with living Dictyostelium discoideum cells migrating through the nanowire array. Cell-induced forces are probed with a resolution of 50 piconewton, while the most flexible nanowires promise to enter the 100 femtonewton realm.

Effect of Loading Method on a Peptide Substrate Reporter in Intact Cells.

Studies of live cells often require loading of exogenous molecules through the cell membrane; however, effects of loading method on experimental results are poorly understood. Therefore, in this work, we compared three methods for loading a fluorescently labeled peptide into cells of the model organism Dictyostelium discoideum. We optimized loading by pinocytosis, electroporation, and myristoylation to maximize cell viability and characterized loading efficiency, localization, and uniformity. We also determined how the loading method affected measurements of enzyme activity on the peptide substrate reporter using capillary electrophoresis. Loading method had a strong effect on the stability and phosphorylation of the peptide. The half-life of the intact peptide in cells was 19 ± 2, 53 ± 15, and 12 ± 1 min, for pinocytosis, electroporation, and myristoylation, respectively. The peptide was phosphorylated only in cells loaded by electroporation. Fluorescence microscopy suggested that the differences between methods were likely due to differences in peptide localization.

Electric Pulses Can Influence Galvanotaxis of Dictyostelium discoideum.

Galvanotaxis, or electrotaxis, plays an essential role in wound healing, embryogenesis, and nerve regeneration. Up until now great efforts have been made to identify the underlying mechanism related to galvanotaxis in various cells under direct current electric field (DCEF) in laboratory studies. However, abundant clinical research shows that non-DCEFs including monopolar or bipolar electric field may also contribute to wound healing and regeneration, although the mechanism remains elusive. Here, we designed a novel electric stimulator and applied DCEF, pulsed DCEF (pDCEF), and bipolar pulse electric field (bpEF) to the cells of Dictyostelium discoideum. The cells had better directional performance under asymmetric 90% duty cycle pDCEF and 80% duty cycle bpEF compared to DCEF, with 10 Hz frequency electric fields eliciting a better cell response than 5 Hz. Interestingly, electrically neutral 50% duty cycle bpEF triggered the highest migration speed, albeit in random directions. The results suggest that electric pulses are vital to galvanotaxis and non-DCEF is promising in both basic and clinical researches.

Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells

The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.

Adenylyl cyclase A mRNA localized at the back of cells is actively translated in live chemotaxing Dictyostelium.

Dictyostelium discoideum cells transport adenylyl cyclase A (ACA)-containing vesicles to the back of polarized cells to relay exogenous cAMP signals during chemotaxis. Fluorescence in situ hybridization (FISH) experiments showed that ACA mRNA is also asymmetrically distributed at the back of polarized cells. By using the MS2 bacteriophage system, we now visualize the distribution of ACA mRNA in live chemotaxing cells. We found that the ACA mRNA localization is not dependent on the translation of the protein product and requires multiple cis-acting elements within the ACA-coding sequence. We show that ACA mRNA is associated with actively translating ribosomes and is transported along microtubules towards the back of cells. By monitoring the recovery of ACA-YFP after photobleaching, we observed that local translation of ACA-YFP occurs at the back of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signals during chemotaxis.

Influence of fast advective flows on pattern formation of Dictyostelium discoideum.

We report experimental and numerical results on pattern formation of self-organizing Dictyostelium discoideum cells in a microfluidic setup under a constant buffer flow. The external flow advects the signaling molecule cyclic adenosine monophosphate (cAMP) downstream, while the chemotactic cells attached to the solid substrate are not transported with the flow. At high flow velocities, elongated cAMP waves are formed that cover the whole length of the channel and propagate both parallel and perpendicular to the flow direction. While the wave period and transverse propagation velocity are constant, parallel wave velocity and the wave width increase linearly with the imposed flow. We also observe that the acquired wave shape is highly dependent on the wave generation site and the strength of the imposed flow. We compared the wave shape and velocity with numerical simulations performed using a reaction-diffusion model and found excellent agreement. These results are expected to play an important role in understanding the process of pattern formation and aggregation of D. discoideum that may experience fluid flows in its natural habitat.