Self-organization of chemoattractant waves inDictyosteliumdepends on F-actin and cell–substrate adhesion

Abstract

In the social amoeba Dictyostelium discoideum, travelling waves of extracellular cyclic adenosine monophosphate (cAMP) self-organize in cell populations and direct aggregation of individual cells to form multicellular fruiting bodies. In contrast to the large body of studies that addressed how movement of cells is determined by spatial and temporal cues encoded in the dynamic cAMP gradients, how cell mechanics affect the formation of a self-generated chemoattractant field has received less attention. Here, we show, by live cell imaging analysis, that the periodicity of the synchronized cAMP waves increases in cells treated with the actin inhibitor latrunculin. Detail analysis of the extracellular cAMP-induced transients of cytosolic cAMP (cAMP relay response) in well-isolated cells demonstrated that their amplitude and duration were markedly reduced in latrunculin-treated cells. Similarly, in cells strongly adhered to a poly-l-lysine-coated surface, the response was suppressed, and the periodicity of the population-level oscillations was markedly lengthened. Our results suggest that cortical F-actin is dispensable for the basic low amplitude relay response but essential for its full amplification and that this enhanced response is necessary to establish high-frequency signalling centres. The observed F-actin dependence may prevent aggregation centres from establishing in microenvironments that are incompatible with cell migration.

Fold-change detection and scale invariance of cell–cell signaling in social amoeba

“…Less is known about adaptation of ACA besides the fact that it depends on receptor phosphorylation (41) and PI3K (16,38). Semirescaling of the response sensitivity has been reported at the level of Ras activation (42), which appears "FCD-like" after population averaging (7,8).…”

“…In general, a response to a step stimulus may not necessarily have the same propensity as that for a slowly varying stimulus. Moreover, directional migration is induced by a traveling-wave stimulus (17,18) and thus may affect the cAMP relay owing to a large overlap in the signal transduction network (16,31,32). To test relevance of the fold-change response property for natural cAMP wave, we used a microfluidics lighthouse (33)-a gradient-generating platform capable of delivering traveling-wave stimulus of various amplitude, frequencies, and speed.…”

“…After prolonged exposure to cAMP, the rise in extracellular cAMP level ceases due to inactivation of adenylyl cyclase (14). As extracellular cAMP level is lowered by degradation, the cells exit from the state of reduced responsivity over the course of several minutes (15,16), and hence the extracellular cAMP level once again starts to elevate. This tendency for the extracellular cAMP level to rise when it is lowered, and to be lowered when it is raised, essentially renders extracellular cAMP level unstable and oscillatory.…”

“…Cells lacking the cAMP-synthesizing enzyme adenylyl cyclase ACA when ectopically forced to differentiate cannot form aggregates unless they are allowed to randomly collide and form cell clusters at high cell densities (23), indicating that robust cell aggregation depends on intercellular cAMP signaling. Recent live-cell imaging studies have elucidated an input-output relation of the cAMP relay response at the single-cell level resolution (16,(24)(25)(26). Although these analyses have implied a role played by the stochastic cAMP relay response at low basal concentrations of extracellular cAMP (∼100 pM) in initiating the synchronized pulses, how they could take place robustly in a wide range of cell densities is yet poorly understood.…”

Tissue self-organization based on collective cell migration by contact activation of locomotion and chemotaxis

“…Purified plasmids were used to transform Dictyostelium cells by electroporation following a standard protocol[53] and selected using 10 μg/mL G418 or 60 μg/mL Hygromycin. Following strains of Dictyostelium discoideum were used for live-cell imaging analysis: AX4 and its derivatives GFP-Lifeact[54]/AX4, Lifeact-mRFPmars[55]/AX4, GFP/ tgrB1 − , tdTomato/ tgrC1 − , GFP-Arp2/AX4, HSPC300-GFP[56]/Lifeact-mRFPmars/AX4, PakBCRIB-mRFP[56]/GFP-Lifeact/AX4, GFP-Lifeact/mRFPmars-Raf1RBD[17]/AX4, GFP-Lifeact/PHcrac-RFP/AX4, pEcmAO-GFP/pD19-RFP/AX4, Epac1-camps[57]/AX4, [tgrB1p]:tgrB1-RFP/ tgrB1 − , [tgrC1p]:tgrC1-GFP/ tgrC1 − , [act15p]:tgrB1-RFP/AX4, [act15p]:tgrC1-GFP/AX4. To obtain pEcmAO-GFP/pD19-RFP/AX4, AX4 cells were co-transformed by electroporation with plasmids pEcmAO-GFP and pD19-RFP.…”