Robustness of Self-Organizing Chemoattractant Field Arising from Precise Pulse Induction of Its Breakdown Enzyme: A Single-Cell Level Analysis of PDE Expression in Dictyostelium

Abstract

The oscillation of chemoattractant cyclic AMP (cAMP) in Dictyostelium discoideum is a collective phenomenon that occurs when the basal level of extracellular cAMP exceeds a threshold and invokes cooperative mutual excitation of cAMP synthesis and secretion. For pulses to be relayed from cell to cell repetitively, secreted cAMP must be cleared and brought down to the subthreshold level. One of the main determinants of the oscillatory behavior is thus how much extracellular cAMP is degraded by extracellular phosphodiesterase (PDE). To date, the exact nature of PDE gene regulation remains elusive. Here, we performed live imaging analysis of mRNA transcripts for pdsA--the gene encoding extracellular PDE. Our analysis revealed that pdsA is upregulated during the rising phase of cAMP oscillations. Furthermore, by analyzing isolated cells, we show that expression of pdsA is strictly dependent on the presence of extracellular cAMP. pdsA is induced only at ∼1 nM extracellular cAMP, which is almost identical to the threshold concentration for the cAMP relay response. The observed precise regulation of PDE expression together with degradation of extracellular cAMP by PDE form a dual positive and negative feedback circuit, and model analysis shows that this sets the cAMP level near the threshold concentration for the cAMP relay response for a wide range of adenylyl cyclase activity. The overlap of the thresholds could allow oscillations of chemoattractant cAMP to self-organize at various starving conditions, making its development robust to fluctuations in its environment.

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“…S1 and SI Materials and Methods). Collective cAMP oscillations occur spontaneously in cell populations continuously perfused with buffer solution at a moderate flow rate (1.5 mL/min), where extracellular cAMP breakdown by secreted phosphodiesterase (PDE) in vivo is effectively emulated by dilution (24,28). First, we tested the effect of adding cAMP in the perfusion flow (hereafter refer to as "exogenous cAMP"), which adds on top of endogenously synthesized and secreted cAMP present in the extracellular space.…”

“…These oscillations are driven intracellularly without the need for clearing of extracellular cAMP-a feature in favor of the Fitzhugh-Nagumo-type scheme. For the population-level oscillations, in contrast, a strict dependency on extracellular cAMP clearing has been demonstrated by a null-mutant of extracellular phosphodiesterase that cannot oscillate unless it is cleared of extracellular cAMP by perfusion (28). It may be that different cAMP receptor forms (52) or receptor types (53) with different binding constants transduce FCD and non-FCD signals.…”

“…Although the FCD mechanism could explain the ability of Dictyostelium cells to execute aggregating behavior at a wide range of cell density (21,22), the present findings do not rule out other mechanisms that may be at work in conjunction. cAMP-dependent regulation of a gene encoding extracellular PDE can provide robustness (28), in this case by keeping the level of extracellular cAMP within a desirable range. Cell-cell heterogeneity in the response sensitivity could also render oscillations robust to cell density change (Fig.…”

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“…By hydrolyzing cAMP at the cell surface, mPDE lowers its local concentration and steepens the gradient; the negative cooperativity of the enzyme helps in this (Nanjundiah & Malchow, ). Masaki et al () have found that in the axenic strain Ax2, external cAMP pulses cause rapid transcription from the pdsA gene. If generally valid, the observation indicates that mPDE activity too rises in parallel with the cAMP signal, which would be another sign of its significance for chemotaxis.…”

“…By hydrolyzing cAMP at the cell surface, mPDE lowers its local concentration and steepens the gradient; the negative cooperativity of the enzyme helps in this (Nanjundiah & Malchow, 1975). Masaki et al (2013) have found that in the axenic strain Ax2, external cAMP pulses cause rapid transcription from the pdsA gene.…”