Satoshi Sawai

The Onset of Collective Behavior in Social Amoebae

All Together Now In the social amoeba Dictyostelium discoideum, periodic synthesis and release of cyclic AMP (cAMP) guides the cellular aggregation required to form fruiting bodies. It has been unclear whether the initiation of this behavior is owing to synchronization of autonomously oscillating cells or whether individual cells remain nonoscillatory unless the entire population becomes oscillatory. Gregor et al. (p. 1021, published online 22 April; see the Perspective by Prindle and Hasty) used live-cell imaging to show that cAMP pulses originate from a specific location in space and that individual cells move in and out of these signaling centers. The observations suggest that oscillations do not originate from autonomous activities of specialized cells. However, individual cells do display stochastic cAMP-pulsing below a threshold external concentration of cAMP, and the generation of synchronized oscillations could only be modeled accurately when this random pulsing was taken into account. Stochastic pulsing of individual cells plays a critical role in initiating cyclic adenosine monophosphate pulses. In the social amoebae Dictyostelium discoideum, periodic synthesis and release of extracellular cyclic adenosine 3′,5′-monophosphate (cAMP) guide cell aggregation and commitment to form fruiting bodies. It is unclear whether these oscillations are an intrinsic property of individual cells or if they exist only as a population-level phenomenon. Here, we showed by live-cell imaging of intact cell populations that pulses originate from a discrete location despite constant exchange of cells to and from the region. In a perfusion chamber, both isolated single cells and cell populations switched from quiescence to rhythmic activity depending on the concentration of extracellular cAMP. A quantitative analysis showed that stochastic pulsing of individual cells below the threshold concentration of extracellular cAMP plays a critical role in the onset of collective behavior.

An autoregulatory circuit for long-range self-organization in Dictyostelium cell populations

Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular structure by chemotaxis, moving towards propagating waves of cyclic AMP that are relayed from cell to cell. Organizing centres are not formed by founder cells, but are dynamic entities consisting of cores of outwardly rotating spiral waves that self-organize in a homogeneous cell population. Spiral waves are ubiquitously observed in chemical reactions as well as in biological systems. Although feedback control of spiral waves in spatially extended chemical reactions has been demonstrated in recent years, the mechanism by which control is achieved in living systems is unknown. Here we show that mutants of the cyclic AMP/protein kinase A pathway show periodic signalling, but fail to organize coherent long-range wave territories, owing to the appearance of numerous spiral cores. A theoretical model suggests that autoregulation of cell excitability mediated by protein kinase A acts to optimize the number of signalling centres.

Phase geometries of two-dimensional excitable waves govern self-organized morphodynamics of amoeboid cells

In both randomly moving Dictyostelium and mammalian cells, phosphatidylinositol (3,4,5)-trisphosphate and F-actin are known to propagate as waves at the membrane and act to push out the protruding edge. To date, however, the relationship between the wave geometry and the patterns of amoeboid shape change remains elusive. Here, by using phase map analysis, we show that morphology dynamics of randomly moving Dictyostelium discoideum cells can be characterized by the number, topology, and position of spatial phase singularities, i.e., points that represent organizing centers of rotating waves. A single isolated singularity near the cellular edge induced a rotational protrusion, whereas a pair of singularities supported a symmetric extension. These singularities appeared by strong phase resetting due to de novo nucleation at the back of preexisting waves. Analysis of a theoretical model indicated excitability of the system that is governed by positive feedback from phosphatidylinositol (3,4,5)-trisphosphate to PI3-kinase activation, and we showed experimentally that this requires F-actin. Furthermore, by incorporating membrane deformation into the model, we demonstrated that geometries of competing waves explain most of the observed semiperiodic changes in amoeboid morphology.

Population fitness and the regulation of Escherichia coli genes by bacterial viruses.

Temperate bacteriophage parasitize their host by integrating into the host genome where they provide additional genetic information that confers higher fitness on the host bacterium by protecting it against invasion by other bacteriophage, by increasing serum resistance, and by coding for toxins and adhesion factors that help the parasitized bacterium invade or evade its host. Here we ask if a temperate phage can also regulate host genes. We find several different host functions that are down-regulated in lysogens. The pckA gene, required for gluconeogenesis in all living systems, is regulated directly by the principal repressor of many different temperate prophage, the cI protein. cI binds to the regulatory region of pckA, thereby shutting down pckA transcription. The pckA regulatory region has target sequences for many other temperate phage repressors, and thus we suggest that down-regulation of the host pckA pathway increases lysogen fitness by lowering the growth rate of lysogens in energy-poor environments, perhaps as an adaptive response to the host predation system or as an aspect of lysogeny that must be offset by down-regulating pckA.

Rectified directional sensing in long-range cell migration

How spatial and temporal information are integrated to determine the direction of cell migration remains poorly understood. Here, by precise microfluidics emulation of dynamic chemoattractant waves, we demonstrate that, in Dictyostelium, directional movement as well as activation of small guanosine triphosphatase Ras at the leading edge is suppressed when the chemoattractant concentration is decreasing over time. This ‘rectification’ of directional sensing occurs only at an intermediate range of wave speed and does not require phosphoinositide-3-kinase or F-actin. From modelling analysis, we show that rectification arises naturally in a single-layered incoherent feedforward circuit with zero-order ultrasensitivity. The required stimulus time-window predicts ~5 s transient for directional sensing response close to Ras activation and inhibitor diffusion typical for protein in the cytosol. We suggest that the ability of Dictyostelium cells to move only in the wavefront is closely associated with rectification of adaptive response combined with local activation and global inhibition.

High-throughput analysis of spatio-temporal dynamics in Dictyostelium

We demonstrate a time-lapse video approach that allows rapid examination of the spatio-temporal dynamics of Dictyostelium cell populations. Quantitative information was gathered by sampling life histories of more than 2,000 mutant clones from a large mutagenesis collection. Approximately 4% of the clonal lines showed a mutant phenotype at one stage. Many of these could be ordered by clustering into functional groups. The dataset allows one to search and retrieve movies on a gene-by-gene and phenotype-by-phenotype basis.

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay

Summary Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Collective oscillations in developing cells: Insights from simple systems

From hormonal secretion to gene expression, multicellular dynamics are rich in oscillatory regulation. When organized in space and time, periodic cell–cell signaling can give rise to long‐range coordination of gene expression and cell movement in tissues. Lack of synchrony of the oscillations on the other hand can serve as a source of initial divergence of cell fate in stem cells. How properties of individual cells can account for collective rhythmic behaviors at the tissue level remains elusive in most cases. Recently, studies in chemical reactions, synthetic gene circuits, yeast and social amoeba Dictyostelium have greatly enhanced our view of collective oscillations in cell populations. From these relatively simple systems, a unified view of how excitable and oscillatory regulations could be tuned and coupled to give rise to tissue‐level oscillations is emerging. The review focuses on recent progress in cyclic adenosine monophosphate oscillations in Dictyostelium and highlights similarities and differences with other systems. We will see that the autonomy of single‐cell level oscillations and different ways in which cells are coupled influence how group‐level information can be encoded in collective oscillations.

SunB, a novel Sad1 and UNC‐84 domain‐containing protein required for development of Dictyostelium discoideum

A gene, sunB, encoding a novel class of Sad1 and UNC‐84 (SUN) domain, was isolated from a cDNA screen for suppressors of a mutation in Dd‐STATa – a Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription). The SunB protein localized in the area around the nucleus in growing cells, but in the multicellular stages it was predominantly found in prespore vacuoles (PSVs). A disruptant of sunB was multinucleated in the vegetative phase; during development it formed mounds with multiple tips and failed to culminate. The mutation was cell autonomous, and showed reduced expression of the prespore marker gene pspA and elevated expression of marker genes for prestalk AB cells. Interestingly, the level of SunB was abnormally high in the prestalk cells of Dd‐STATa mutants, which are defective in culmination. We conclude that SunB is essential for accurate prestalk/prespore differentiation during Dictyostelium development and that its cell‐type dependent localization is regulated by a Dd‐STATa‐mediated signaling pathway.

A Design Principle of Group-level Decision Making in Cell Populations

Populations of cells often switch states as a group to cope with environmental changes such as nutrient availability and cell density. Although the gene circuits that underlie the switches are well understood at the level of single cells, the ways in which such circuits work in concert among many cells to support group-level switches are not fully explored. Experimental studies of microbial quorum sensing show that group-level changes in cellular states occur in either a graded or an all-or-none fashion. Here, we show through numerical simulations and mathematical analysis that these behaviors generally originate from two distinct forms of bistability. The choice of bistability is uniquely determined by a dimensionless parameter that compares the synthesis and the transport of the inducing molecules. The role of the parameter is universal, such that it not only applies to the autoinducing circuits typically found in bacteria but also to the more complex gene circuits involved in transmembrane receptor signaling. Furthermore, in gene circuits with negative feedback, the same dimensionless parameter determines the coherence of group-level transitions from quiescence to a rhythmic state. The set of biochemical parameters in bacterial quorum-sensing circuits appear to be tuned so that the cells can use either type of transition. The design principle identified here serves as the basis for the analysis and control of cellular collective decision making.