Modelling cell movement, cell differentiation, cell sorting and proportion regulation in Dictyostelium discoideum aggregations.

Understanding the mechanisms that control tissue morphogenesis and homeostasis is a central goal not only in developmental biology but also has great relevance for our understanding of various diseases, including cancer. A model organism that is widely used to study the control of tissue morphogenesis and proportioning is the Dictyostelium discoideum. While there are mathematical models describing the role of chemotactic cell motility in the Dictyostelium assembly and morphogenesis of multicellular tissues, as well as models addressing possible mechanisms of proportion regulation, there are no models incorporating both these key aspects of development. In this paper, we introduce a 1D hyperbolic model to investigate the role of two morphogens, DIF and cAMP, on cell movement, cell sorting, cell-type differentiation and proportioning in Dictyostelium discoideum. First, we use the non-spatial version of the model to study cell-type transdifferentiation. We perform a steady-state analysis of it and show that, depending on the shape of the differentiation rate functions, multiple steady-state solutions may occur. Then we incorporate spatial dynamics into the model, and investigate the transdifferentiation and spatial positioning of cells inside the newly formed structures, following the removal of prestalk or prespore regions of a Dictyostelium slug. We show that in isolated prespore fragments, a tipped mound-like aggregate can be formed after a transdifferentiation from prespore to prestalk cells and following the sorting of prestalk cells to the centre of the aggregate. For isolated prestalk fragments, we show the formation of a slug-like structure containing the usual anterior-posterior pattern of prestalk and prespore cells.

Alignment of an acoustic manipulation device with cepstral analysis of electronic impedance data.

Acoustic particle manipulation is an emerging technology that uses ultrasonic standing waves to position objects with pressure gradients and acoustic radiation forces. To produce strong standing waves, the transducer and the reflector must be aligned properly such that they are parallel to each other. This can be a difficult process due to the need to visualise the ultrasound waves and as higher frequencies are introduced, this alignment requires higher accuracy. In this paper, we present a method for aligning acoustic resonators with cepstral analysis. This is a simple signal processing technique that requires only the electrical impedance measurement data of the resonator, which is usually recorded during the fabrication process of the device. We first introduce the mathematical basis of cepstral analysis and then demonstrate and validate it using a computer simulation of an acoustic resonator. Finally, the technique is demonstrated experimentally to create many parallel linear traps for 10 µm fluorescent beads inside an acoustic resonator.

Emergent cell and tissue dynamics from subcellular modeling of active biomechanical processes.

Cells and the tissues they form are not passive material bodies. Cells change their behavior in response to external biochemical and biomechanical cues. Behavioral changes, such as morphological deformation, proliferation and migration, are striking in many multicellular processes such as morphogenesis, wound healing and cancer progression. Cell-based modeling of these phenomena requires algorithms that can capture active cell behavior and their emergent tissue-level phenotypes. In this paper, we report on extensions of the subcellular element model to model active biomechanical subcellular processes. These processes lead to emergent cell and tissue level phenotypes at larger scales, including (i) adaptive shape deformations in cells responding to slow stretching, (ii) viscous flow of embryonic tissues, and (iii) streaming patterns of chemotactic cells in epithelial-like sheets. In each case, we connect our simulation results to recent experiments.

A 'chemotactic dipole' mechanism for large-scale vortex motion during primitive streak formation in the chick embryo.

Primitive streak formation in the chick embryo involves significant coordinated cell movement lateral to the streak, in addition to the posterior-anterior movement of cells in the streak proper. Cells lateral to the streak are observed to undergo 'polonaise movements', i.e. two large counter-rotating vortices, reminiscent of eddies in a fluid. In this paper, we propose a mechanism for these movement patterns which relies on chemotactic signals emitted by a dipolar configuration of cells in the posterior region of the epiblast. The 'chemotactic dipole' consists of adjacent regions of cells emitting chemo-attractants and chemo-repellents. We motivate this idea using a mathematical analogy between chemotaxis and electrostatics, and test this idea using large-scale computer simulations. We implement active cell response to both neighboring mechanical interactions and chemotactic gradients using the Subcellular Element Model. Simulations show the emergence of large-scale vortices of cell movement. The length and time scales of vortex formation are in reasonable agreement with experimental data. We also provide quantitative estimates for the robustness of the chemotaxis dipole mechanism, which indicate that the mechanism has an error tolerance of about 10% to variation in chemotactic parameters, assuming that only 1% of the cell population is involved in emitting signals. This tolerance increases for larger populations of cells emitting signals.

Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN.

PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN's lipid phosphatase activity in regulating the PI3K signalling pathway is recognized, the significance of PTEN's other mechanisms of action is currently unclear. In this study, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant, we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells, the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP(3) levels or the downstream protein kinase Akt/PKB. Finally, in three-dimensional Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provide a novel tool to address the significance of PTEN's separable lipid and protein phosphatase activities and suggest that both activities suppress proliferation and together suppress invasion.

The regulation of cell migration by PTEN.

In vertebrates, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) regulates many cellular processes through its PtdIns(3,4,5)P3 lipid phosphatase activity, antagonizing PI3K (phosphoinositide 3-kinase) signalling. Given the important role of PI3Ks in the regulation of directed cell migration and the role of PTEN as an inhibitor of migration, it is somewhat surprising that data now indicate that PTEN is able to regulate cell migration independent of its lipid phosphatase activity. Here, we discuss the role of PTEN in the regulation of cell migration.

Becoming Multicellular by Aggregation; The Morphogenesis of the Social Amoebae Dicyostelium discoideum.

The organisation and form of most organisms is generated during theirembryonic development and involves precise spatial and temporal controlof cell division, cell death, cell differentiation and cell movement.Differential cell movement is a particularly important mechanism in thegeneration of form. Arguably the best understood mechanism of directedmovement is chemotaxis. Chemotaxis plays a major role in the starvationinduced multicellular development of the social amoebae Dictyostelium.Upon starvation up to 10(5) individual amoebae aggregate to form afruiting body. In this paper we review the evidence that the movement ofthe cells during all stages of Dictyostelium development is controlled bypropagating waves of cAMP which control the chemotactic movement ofthe cells. We analyse the complex interactions between cell-cell signallingresulting in cAMP waves of various geometries and cell movement whichresults in a redistribution of the signalling sources and therefore changes thegeometry of the waves. We proceed to show how the morphogenesis,including aggregation stream and mound formation, slug formation andmigration, of this relatively simple organism is beginning to be understoodat the level of rules for cell behaviour, which can be tested experimentallyand theoretically by model calculations.

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Propagating chemoattractant waves coordinate periodic cell movement in Dictyostelium slugs.

Migration and behaviour of Dictyostelium slugs results from coordinated movement of its constituent cells. It has been proposed that cell movement is controlled by propagating waves of cAMP as during aggregation and in the mound. We report the existence of optical density waves in slugs; they are initiated in the tip and propagate backwards. The waves reflect periodic cell movement and are mediated by cAMP, as injection of cAMP or cAMP phosphodiesterase disrupts wave propagation and results in effects on cell movement and, therefore, slug migration. Inhibiting the function of the cAMP receptor cAR1 blocks wave propagation, showing that the signal is mediated by cAR1. Wave initiation is strictly dependent on the tip; in decapitated slugs no new waves are initiated and slug movement stops until a new tip regenerates. Isolated tips continue to migrate while producing waves. We conclude from these observations that the tip acts as a pacemaker for cAMP waves that coordinate cell movement in slugs.

cAMP receptor affinity controls wave dynamics, geometry and morphogenesis in Dictyostelium.

Serpentine G-protein-coupled cAMP receptors are key components in the detection and relay of the extracellular cAMP waves that control chemotactic cell movement during Dictyostelium development. During development the cells sequentially express four closely related cAMP receptors of decreasing affinity. In this study, we investigated the effect of cAMP receptor type and affinity on the dynamics of cell-cell signalling in vivo, by measuring the dynamics of wave initiation and propagation in a variety of cAMP receptor mutants. We found that receptor affinity controls the frequency of wave initiation, but it does not determine wave propagation velocity, thus resulting in dramatic changes in wave geometry. In the limiting case, the affinity of the receptor is so low that waves can still be initiated but no stable centres form - thus, the cells cannot aggregate. In mounds, expression of low affinity receptors results in slow concentric waves instead of the normally observed multi-armed spiral waves. Under these conditions there is no rotational cell movement and the hemispherical mounds cannot transform into slugs. These results highlight the importance of receptor number and affinity in the proper control of cell-cell signalling dynamics required for the successful completion of development.

Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation.

Dd-STATa, the Dictyostelium STAT (signal transducer and activator of transcription) protein, is selectively localised in the nuclei of a small subset of prestalk cells located in the slug tip. Injection of cAMP into the extracellular spaces in the rear of the slug induces rapid nuclear translocation of a Dd-GFP:STATa fusion protein in prespore cells surrounding the site of injection. This suggests that cAMP signals that emanate from the tip direct the localised nuclear accumulation of Dd-STATa. It also shows that prespore cells are competent to respond to cAMP, by Dd-STATa activation, and it implies that cAMP signalling is in some way limiting in the rear of the slug. Co-injection of a specific inhibitor of the cAR1 serpentine cAMP receptor almost completely prevents the cAMP-induced nuclear translocation, showing that most or all of the cAMP signal is transduced by cAR1. Dd-GFP:STATa also rapidly translocates into the nuclei of cells adjoining the front and back cut edges when a slug is bisected. Less severe mechanical disturbances, such as pricking the rear of a slug with an unfilled micropipette, also cause a more limited nuclear translocation of Dd-GFP:STATa. We propose that these signalling events form part of a repair mechanism that is activated when the migrating slug suffers mechanical damage.

The control of chemotactic cell movement during Dictyostelium morphogenesis.

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium.

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.

Modeling chemotactic cell sorting during Dictyostelium discoideum mound formation.

Coordinated cell movement is a major mechanism of the multicellular development of most organisms. The multicellular morphogenesis of the slime mould Dictyostelium discoideum, from single cells into a multicellular fruiting body, results from differential chemotactic cell movement. During aggregation cells differentiate into prestalk and prespore cells that will form the stalk and spores in the fruiting body. These cell types arise in a salt and pepper pattern after what the prestalk cells chemotactically sort out to form a tip. The tip functions as an organizer because it directs the further development. It has been difficult to get a satisfactory formal description of the movement behavior of cells in tissues. Based on our experiments, we consider the aggregate as a drop of a viscous fluid and show that this consideration is very well suited to mathematically describe the motion of cells in the tissue. We show that the transformation of a hemispherical mound into an elongated slug can result from the coordinated chemotactic cell movement in response to scroll waves of the chemoattractant cAMP. The model calculations furthermore show that cell sorting can result from differences in chemotactic cell movement and cAMP relay kinetics between the two cell types. During this process, the faster moving and stronger signaling cells collect on the top of the mound to form a tip. The mound then extends into an elongated slug just as observed in experiments. The model is able to describe cell movement patterns in the complex multicellular morphogenesis of Dictyostelium rather well and we expect that this approach may be useful in the modeling of tissue transformations in other systems.

Morphogenetic cell movement in Dictyostelium.

Dictyostelium morphogenesis starts with the chemotactic aggregation of starving individual cells. The cells move in response to propagating waves of the chemoattractant cyclic AMP initiated by cells in the aggregation centre. During aggregation the cells begin to differentiate into several types with different signalling and chemotactic properties. These cell types sort out from each other to form an axial pattern in the slug. There is now good evidence that periodic chemotactic signals not only control aggregation, but also later stages of morphogenesis. These signals take the form of target patterns, spirals, multi-armed spirals and scroll waves. I will discuss their role in the control of cell movement during mound and slug formation and in the formation of the fruiting body.

Propagating waves control Dictyostelium discoideum morphogenesis.

The morphogenesis of Dictyostelium results from the coordinated movement of starving cells to form a multicellular aggregate (mound) which transforms into a motile slug and finally a fruiting body. Cells differentiate in the mound and sort out to form an organised pattern in the slug and fruiting body. During aggregation, cell movement is controlled by propagating waves of the chemo-attractant cAMP. We show that mounds are also organised by propagating waves. Their geometry changes from target or single armed spirals during aggregation to multi-armed spiral waves in the mound. Some mounds develop transiently into rings in which multiple propagating wave fronts can still be seen. We model cell sorting in the mound stage assuming cell type specific differences in cell movement speed and excitability. This sorting feeds back on the wave geometry to generate twisted scroll waves in the slug. Slime mould morphogenesis can be understood in terms of wave propagation directing chemotactic cell movement.

Induction of optical density waves and chemotactic cell movement in Dictyostelium discoideum by microinjection of cAMP pulses.

The development of most multicellular organisms involves coordinated cell movement. The early aggregation of Dictyostelium cells has been shown to be mediated by chemotactic movement to propagating waves of cAMP. We have proposed that propagating waves of a chemoattractant, most likely cAMP, also control the movement of cells in mounds and slugs. We have now used periodic pressure injection of pulses of cAMP in the extracellular space of aggregation streams, mounds, and slugs to investigate whether these signals can be relayed and control cell movement, using quantitative digital time-lapse microscopy. Our major findings are (1) short (0.1 s) pulses of cAMP (10(7) molecules) were able to elicit optical density (OD) waves in fields of aggregating amoebae. They propagate from the micropipet outward and interact with endogenous OD waves. (2) Periodic injection of cAMP pulses into aggregation streams blocked the pulses coming from the center and led to the rapid accumulation of cells downstream of the pipet around the pipet. (3) Injection of pulses of cAMP into mounds elicited OD waves, which propagated from the pipet outward and interacted with the endogenous waves, indicating that the same propagator carries them. (4) Periodic microinjection of cAMP in the prespore zone of slugs led to accumulation of anterior-like cells around the micropipet followed by tip formation. Furthermore, the cAMP signal could control the spacing of the endogenous sorting pattern. These results strongly support the hypothesis that the optical density waves observed during early development up to the mound stage represent propagating cAMP waves. They suggest furthermore that cAMP is the morphogen that controls cell movements in slugs.

Analysis of cell movement and signalling during ring formation in an activated G alpha1 mutant of Dictyostelium discoideum that is defective in prestalk zone formation.

Mound formation in the cellular slime mould Dictyostelium results from the chemotactic aggregation of competent cells. Periodic cAMP signals propagate as multiarmed spiral waves and coordinate the movement of the cells. In the late aggregate stage the cells differentiate into prespore and several prestalk cell types. Prestalk cells sort out chemotactically to form the tip, which then controls all further development. The tip organises cell movement via a scroll wave that converts to planar waves in the prespore zone leading to rotational cell movement in the tip and periodic forward movement in the prespore zone. Expression of an activated G alpha1 protein under its own promoter leads to a severely altered morphogenesis from the mound stage onwards. Instead of forming a tipped mound, the cells form a ring-shaped structure without tip. Wave propagation pattern and dynamics during aggregation and mound formation in the mutant are indistinguishable from the parental strain AX3. However, at the time of tip formation the spiral waves that organise the late aggregate do not evolve in a scroll-organising centre in the tip but transform into a circularly closed (twisted) scroll ring wave. This leads to the formation of a doughnut-shaped aggregate. During further development, the doughnut increases in diameter and the twisted scroll wave converts into a train of planar waves, resulting in periodic rotational cell movement. Although biochemical consequences resulting from this mutation are still unclear, it must affect prestalk cell differentiation. The mutant produces the normal proportion of prespore cells but is unable to form functional prestalk cells, i.e., prestalk cells with an ability to sort out from the prespore cells and form a prestalk zone. Failure of sorting leads to an altered signal geometry, ring-shaped scroll waves, that then directs ring formation. This mutant demonstrates the importance of prestalk cell sorting for the stabilisation of the scroll wave that organises the tip.

Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates.

Extracellular cAMP is a critical messenger in the multicellular development of the cellular slime mold Dictyostelium discoideum. The levels of cAMP are controlled by a cyclic nucleotide phosphodiesterase (PDE) that is secreted by the cells. The PDE gene (pdsA) is controlled by three promoters that permit expression during vegetative growth, during aggregation, and in prestalk cells of the older structures. Targeted disruption of the gene aborts development, and complementation with a modified pdsA restores development. Two distinct promoters must be used for full complementation, and an inhibitory domain of the PDE must be removed. We took advantage of newly isolated PDE-null cells and the natural chimerism of the organism to ask whether the absence of PDE affected individual cell behavior. PDE-null cells aggregated with isogenic wild-type cells in chimeric mixtures, but could not move in a coordinated manner in mounds. The wild-type cells move inward toward the center of the mound, leaving many of the PDE-null cells at the periphery of the aggregate. During the later stages of development, PDE-null cells in the chimera segregate to regions which correspond to the prestalk region and the rear of the slug. Participation in the prespore/spore population returns with the restoration of a modified pdsA to the null cells.

Periodic phenomena in Proteus mirabilis swarm colony development.

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.