Dictyostelium Discoideum Cells Research Articles

Cells responding to chemoattractant on a structured substrate

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.

Role of the Dimerization Domain of Filamin in Dictyostelium discoideum Response to Shear Flow

Molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation, which is critical in homeostasis and many diseases, are not well understood. Dictyostelium discoideum cells exposed to a brief burst of shear flow show rapid and transient activation of multiple components of the signal transduction network that participates in directed migration of these cells. Previous data from our laboratory demonstrated that actin crosslinking protein filamin is involved in the ability of cells to respond to shear flow. We also found that the actin‐binding domain is required for filamin’s function in this context. To determine if the dimerization domain (DD) is also required for filamin’s role in sensing/transmitting mechanical stimuli we generated a truncation construct of filamin lacking DD (FLNΔDD) and expressed it in wild‐type or filamin‐null cells. We found that FLNΔDDwas able to relocalize to the cortex of both wild‐type and filamin‐null cells following 2‐sec stimulation with shear flow, suggesting that dimerization between filamin molecules is not required for their recruitment to the cortex. To detect activation of the signal transduction network in the presence or absence of FLNΔDD, we used fluorescently‐tagged Ras binding domain biosensor that detects active Ras and was previously shown to relocalize to the cortex following mechanical stimulation. Surprisingly, FLNΔDDwas able to rescue the reduced response of filamin‐null cells to shear flow stimulation, suggesting that dimerization of filamin may not be needed for its ability to sense or transmit mechanical cues. However, the presence of FLNΔDD may also alter the overall organization of the cytoskeletal network, potentially explaining changes in mechanotransduction. We are currently investigating these possibilities.

A PKC that controls polyphosphate levels, pinocytosis and exocytosis, regulates stationary phase onset in Dictyostelium.

Many cells can pause their growth cycle, a topic much enriched by studies of the stationary phase (SP) of model microorganisms. Although several kinases are implicated in SP onset, whether protein kinase C has a role remains unknown. We show that Dictyostelium discoideum cells lacking pkcA entered SP at a reduced cell density, but only in shaking conditions. Precocious SP entry occurs because levels of extracellular polyphosphate (polyP) reach the threshold needed to induce the SP onset at a lower cell density than seen in wild-type cells; adding exopolyphosphatase to pkcA- cells reverses the effect and mimics wild-type growth. PkcA-mediated regulation of polyP depended on inositol hexakisphosphate kinase and phospholipase D. PkcA- mutants also had higher F-actin levels, higher rates of exocytosis and lower pinocytosis rates. Postlysosomes were smaller and present in fewer pkcA- cells compared to the wild type. Overall, the results suggest that a reduced PkcA level triggers SP primarily because cells do not acquire or retain nutrients as efficiently, thus mimicking, or amplifying, the conditions of actual starvation. This article has an associated First Person interview with the first author of the paper.

A role for myosin II clusters and membrane energy in cortex rupture for Dictyostelium discoideum.

Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum, white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cortex in locations where blebs are expected to form. In this work, we examine the role of membrane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-agarose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at positions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy.

From Single to Collective Motion of Social Amoebae: A Computational Study of Interacting Cells

The coupling of the internal mechanisms of cell polarization to cell shape deformations and subsequent cell crawling poses many interdisciplinary scientific challenges. Several mathematical approaches have been proposed to model the coupling of both processes, where one of the most successful methods relies on a phase field that encodes the morphology of the cell, together with the integration of partial differential equations that account for the polarization mechanism inside the cell domain as defined by the phase field. This approach has been previously employed to model the motion of single cells of the social amoeba Dictyostelium discoideum, a widely used model organism to study actin-driven motility and chemotaxis of eukaryotic cells. Besides single cell motility, Dictyostelium discoideum is also well-known for its collective behavior. Here, we extend the previously introduced model for single cell motility to describe the collective motion of large populations of interacting amoebae by including repulsive interactions between the cells. We performed numerical simulations of this model, first characterizing the motion of single cells in terms of their polarity and velocity vectors. We then systematically studied the collisions between two cells that provided the basic interaction scenarios also observed in larger ensembles of interacting amoebae. Finally, the relevance of the cell density was analyzed, revealing a systematic decrease of the motility with density, associated with the formation of transient cell clusters that emerge in this system even though our model does not include any attractive interactions between cells. This model is a prototypical active matter system for the investigation of the emergent collective dynamics of deformable, self-driven cells with a highly complex, nonlinear coupling of cell shape deformations, self-propulsion and repulsive cell-cell interactions. Understanding these self-organization processes of cells like their autonomous aggregation is of high relevance as collective amoeboid motility is part of wound healing, embryonic morphogenesis or pathological processes like the spreading of metastatic cancer cells.

Actin-myosin dynamics during bleb stabilization

Blebs are pressure driven spherical protrusions of the plasma membrane which form when a patch of cell membrane is detached from the cortex and expands under the influence of flowing cytosol. The stabilization of the membrane protrusion is characterized by a complete degradation of the old cortex (actin scar) and the formation of a new cortex beneath the protruded membrane, a process that takes about 20 seconds in Dictyostelium Discoideum cells. The mechanism by which the cortex is degraded at the actin scar and simultaneously reformed at the developing bleb boundary is not fully understood.

Confirmation and variability of the Allee effect in Dictyostelium discoideum cell populations, possible role of chemical signaling within cell clusters

In studies of the unicellular eukaryote Dictyostelium discoideum, many have anecdotally observed that cell dilution below a certain ‘threshold density’ causes cells to undergo a period of slow growth (lag). However, little is documented about the slow growth phase and the reason for different growth dynamics below and above this threshold density. In this paper, we extend and correct our earlier work to report an extensive set of experiments, including the use of new cell counting technology, that set this slow-to-fast growth transition on a much firmer biological basis. We show that dilution below a certain density (around 104 cells ml−1) causes cells to grow slower on average and exhibit a large degree of variability: sometimes a sample does not lag at all, while sometimes it takes many moderate density cell cycle times to recover back to fast growth. We perform conditioned media experiments to demonstrate that a chemical signal mediates this endogenous phenomenon. Finally, we argue that while simple models involving fluid transport of signal molecules or cluster-based signaling explain typical behavior, they do not capture the high degree of variability between samples but nevertheless favor an intra-cluster mechanism.

Weak solutions to a class of signal-dependent motility Keller-Segel systems with superlinear damping

This paper is concerned with a class of signal-dependent motility Keller-Segel systems{ut=∇⋅(D(v)∇u−S(v)u∇v)+f(u),x∈Ω,t>0,vt=Δv−v+u,x∈Ω,t>0, under no-flux boundary conditions in a smooth bounded domain Ω⊂Rn. This model describes the aggregation phase of Dictyostelium discoideum cells in response to the secreted chemical signal cyclic adenosine monophosphate. The parameter functions D and S satisfyD,S∈C3((0,+∞)), and there exist constants k,K,x0>0 such thatk≤D(s)≤K,|S(s)|≤Kfor alls∈(x0,+∞). The generalized logistic source f∈C1([0,+∞)) satisfies f(0)=0 and there exists l>max⁡{2(n+2)n+4,2(n−1)n} such thatlims→+∞⁡f(s)sl=−∞. The main result of the paper is to show that the existence of the global-in-time solution in an appropriately generalized sense to the model for all sufficiently smooth initial data which indicates that superlinear damping can rule out blow-up for such kind of models.

Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion

Motile cells can use and switch between different modes of migration. Here, we use traction force microscopy and fluorescent labeling of actin and myosin to quantify and correlate traction force patterns and cytoskeletal distributions in Dictyostelium discoideum cells that move and switch between keratocyte‐like fan‐shaped, oscillatory, and amoeboid modes. We find that the wave dynamics of the cytoskeletal components critically determine the traction force pattern, cell morphology, and migration mode. Furthermore, we find that fan‐shaped cells can exhibit two different propulsion mechanisms, each with a distinct traction force pattern. Finally, the traction force patterns can be recapitulated using a computational model, which uses the experimentally determined spatiotemporal distributions of actin and myosin forces and a viscous cytoskeletal network. Our results suggest that cell motion can be generated by friction between the flow of this network and the substrate.

Mathematical modeling of chemotaxis guided amoeboid cell swimming

Cells and microorganisms adopt various strategies to migrate in response to different environmental stimuli. To date, many modeling research has focused on the crawling-based Dictyostelium discoideum (Dd) cells migration induced by chemotaxis, yet recent experimental results reveal that even without adhesion or contact to a substrate, Dd cells can still swim to follow chemoattractant signals. In this paper, we develop a modeling framework to investigate the chemotaxis induced amoeboid cell swimming dynamics. A minimal swimming system consists of one deformable Dd amoeboid cell and a dilute suspension of bacteria, and the bacteria produce chemoattractant signals that attract the Dd cell. We use the mathematical amoeba model to generate Dd cell deformation and solve the resulting low Reynolds number flows, and use a moving mesh based finite volume method to solve the reaction–diffusion–convection equation. Using the computational model, we show that chemotaxis guides a swimming Dd cell to follow and catch bacteria, while on the other hand, bacterial rheotaxis may help the bacteria to escape from the predator Dd cell.

On the parabolic‐elliptic Keller–Segel system with signal‐dependent motilities: A paradigm for global boundedness and steady states

This paper is concerned with a parabolic‐elliptic Keller–Segel system where both diffusive and chemotactic coefficients (motility functions) depend on the chemical signal density. This system was originally proposed by Keller and Segel to describe the aggregation phase of Dictyostelium discoideum cells in response to the secreted chemical signal cyclic adenosine monophosphate (cAMP), but the available analytical results are very limited by far. Considering system in a bounded smooth domain with Neumann boundary conditions, we establish the global boundedness of solutions in any dimensions with suitable general conditions on the signal‐dependent motility functions, which are applicable to a wide class of motility functions. The existence/nonexistence of non‐constant steady states is studied and abundant stationary profiles are found. Some open questions are outlined for further pursues. Our results demonstrate that the global boundedness and profile of stationary solutions to the Keller–Segel system with signal‐dependent motilities depend on the decay rates of motility functions, space dimensions and the relation between the diffusive and chemotactic motilities, which makes the dynamics immensely wealthy.

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.

Global Boundedness of the Fully Parabolic Keller-Segel System with Signal-Dependent Motilities

This paper establishes the global uniform-in-time boundedness of solutions to the following Keller-Segel system with signal-dependent diffusion and chemotaxis $$ \left \{ \textstyle\begin{array}{l@{\quad }l} u_{t}=\nabla \cdot (\gamma (v)\nabla u-u\phi (v)\nabla v),\quad & x \in \Omega , t>0, \\ \displaystyle { v_{t}=d\Delta v- v+u},\quad & x\in \Omega , t>0 \\ \end{array}\displaystyle \right . $$ in a bounded domain \(\Omega \subset \mathbb{R}^{N}(N\leq 4)\) with smooth boundary, where the density-dependent motility functions \(\gamma (v)\) and \(\phi (v)\) denote the diffusive and chemotactic coefficients, respectively. The model was originally proposed by Keller and Segel in (J. Theor. Biol. 30:225–234, 1970) to describe the aggregation phase of Dictyostelium discoideum cells, where the two motility functions satisfy a proportional relation \(\phi (v)=(\alpha -1)\gamma '(v)\) with \(\alpha >0\) denoting the ratio of effective body length (i.e. distance between receptors) to the step size. The major technical difficulty in the analysis is the possible degeneracy of diffusion. In this work, we show that if \(\gamma (v)>0\) and \(\phi (v)>0\) are smooth on \([0,\infty )\) and satisfy $$ \inf _{v\geq 0} \frac{d\gamma (v)}{v\phi (v)(v\phi (v)+d-\gamma (v))_{+}}>\frac{N}{2}, $$ then the above Keller-Segel system subject to Neumann boundary conditions admits classical solutions uniformly bounded in time. The main idea of proving our results is the estimates of a weighted functional \(\int _{\Omega }u^{p}v^{-q}dx\) for \(p>\frac{N}{2}\) by choosing a suitable exponent \(p\) depending on the unknown \(v\), by which we are able to derive a uniform \(L^{\infty }\)-norm of \(v\) and hence rule out the diffusion degeneracy.

Physical Control of Intracellular Waves with Nanotopography and Electric Fields

Self-organized waves of coupled signal transduction and cytoskeletal activities have been observed in many cell types, and both experimental and simulation studies show that these intracellular waves drive protrusions and regulate random migration of cells. Here we investigate how the wave system responds to physical external cues - electric fields (EFs) and nanotopography. We use electrofused giant Dictyostelium discoideum cells, which provides a large spatial extent for wave dynamics in a single cell, for exploring the system's local response to external signals. We show that nanotopography leads to quasi one-dimensional waves along topographic features, and that EFs bias the preferred location of wave initiation, and guide wave propagation. Our work reveals that EFs, as an external input, can influence the intracellular wave properties and change the thresholds of activation of the excitable network. This work is funded by the Air Force Office of Scientific Research grant FA9550-16-1-0052.

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.

Ceramide Analysis by Multiple Linked-Scan Mass Spectrometry Using a Tandem Quadrupole Instrument.

Ceramides are a special class of sphingolipids and play a central role in sphingolipid metabolism, and have diverse structures. In this book chapter, tandem quadrupole mass spectrometric approaches applying multiple linked scannings including various constant neutral loss scan (NLS) and precursor ion scan (PIS), the unique applicable feature of a triple-stage quadrupole (TSQ) instrument for analysis of ceramides desorbedas [M-H]- and [M+Li]+ ions are described. Thesemultiple dimensional tandem mass spectrometric approaches are fully adapted to the conventional shotgun lipidomics workflow with minimal or without prior chromatographic separation to profile ceramide molecules, and thus detection of a whole class of ceramide or various specific ceramide subclasses in crude lipid extractcan be achieved. With addition of internal standard(s), semi-quantitation of ceramide in the lipid extract of biological origin is possible. Examples have shown promise in ceramide profiling of several whole lipid extracts from porcine brain, the model Dictyostelium Discoideum cells for cancer study, and skin.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron‐electron resonance (DEER) with site‐directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin‐label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two‐armed GdIII complexes, GdIII‐CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

Analysis of barotactic and chemotactic guidance cues on directional decision-making of Dictyostelium discoideum cells in confined environments

Neutrophils and dendritic cells when migrating in confined environments have been shown to actuate a directional choice toward paths of least hydraulic resistance (barotaxis), in some cases overriding chemotactic responses. Here, we investigate whether this barotactic response is conserved in the more primitive model organism Dictyostelium discoideum using a microfluidic chip design. This design allowed us to monitor the behavior of single cells via live imaging when confronted with bifurcating microchannels, presenting different combinations of hydraulic and chemical stimuli. Under the conditions employed we find no evidence in support of a barotactic response; the cells base their directional choices on the chemotactic cues. When the cells are confronted by a microchannel bifurcation, they often split their leading edge and start moving into both channels, before a decision is made to move into one and retract from the other channel. Analysis of this decision-making process has shown that cells in steeper nonhydrolyzable adenosine- 3', 5'- cyclic monophosphorothioate, Sp- isomer (cAMPS) gradients move faster and split more readily. Furthermore, there exists a highly significant strong correlation between the velocity of the pseudopod moving up the cAMPS gradient to the total velocity of the pseudopods moving up and down the gradient over a large range of velocities. This suggests a role for a critical cortical tension gradient in the directional decision-making process.

Cellular velocity, electrical persistence and sensing in developed and vegetative cells during electrotaxis.

Cells have the ability to detect electric fields and respond to them with directed migratory movement. Investigations identified genes and proteins that play important roles in defining the migration efficiency. Nevertheless, the sensing and transduction mechanisms underlying directed cell migration are still under discussion. We use Dictyostelium discoideum cells as model system for studying eukaryotic cell migration in DC electric fields. We have defined the temporal electric persistence to characterize the memory that cells have in a varying electric field. In addition to imposing a directional bias, we observed that the electric field influences the cellular kinematics by accelerating the movement of cells along their paths. Moreover, the study of vegetative and briefly starved cells provided insight into the electrical sensing of cells. We found evidence that conditioned medium of starved cells was able to trigger the electrical sensing of vegetative cells that would otherwise not orient themselves in the electric field. This observation may be explained by the presence of the conditioned medium factor (CMF), a protein secreted by the cells, when they begin to starve. The results of this study give new insights into understanding the mechanism that triggers the electrical sensing and transduces the external stimulus into directed cell migration. Finally, the observed increased mobility of cells over time in an electric field could offer a novel perspective towards wound healing assays.

You can find your own way

Cell Biology Migration of cells through tissues and embryos is often steered by gradients of attractive chemicals in a process called chemotaxis. Cells are best at navigating complex routes, for which they use “self-generated chemotaxis” and create their own attractant gradients. An example of this is when neutrophils migrate into tissues to attack infection. Using modeling and live-cell data, Tweedy et al. found that self-generated chemotaxis allows cells to obtain surprising amounts of information about their environment. Cells of the slime mold Dictyostelium discoideum and mouse pancreatic cancer–derived cells were able to use the diffusion of attractants to identify the best route through complex mazes, even when the correct path was long and twisted, without ever entering incorrect paths. Science , this issue p. [eaay9792][1] [1]: /lookup/doi/10.1126/science.aay9792