Abstracts from contributions to recent international Dictyostelium meeting in Tsukuba/Japan

Currently, I am in the process of finishing up my work on amoeba signaling and aggregation in Tokyo. This work is in collaboration with and in the laboratory of Satoshi Sawai at the University of Tokyo. We also collaborate with Koichi Fujimoto, a theorist at the same institution. While no papers have been published yet, as a preview pasted below are three abstracts of contributions to a recent international Dictyostelium meeting held in Tsukuba/Japan.

A phase transition to collective behavior in Dictyostelium cell populations

Collective dynamics are widely observed during development of multicellular bodies and emerge as a result of communication among individual cells via signaling molecules. However, little is known experimentally of the fundamental features that describe how the highly nonlinear spatio-temporal dynamics at the single-cell level can give rise to coherent dynamics at the population level. The prototypical example is cell-to-cell signaling among social amoebae Dictyostelium, where a few hundred thousand cells aggregate to form a fruiting body. cAMP is synthesized and secreted periodically and serves as a cue that directs chemotaxis of individual cells. Here we use a FRET-based sensor protein, combined with live-imaging, to monitor cytosolic cAMP levels in developing Dictyostelium cells. Single cell resolution timelapse recordings of cell populations during the first 10 hours of development reveal the very onset of periodic, spike-like signaling, as well as sequential changes in the signaling frequency. The input-output relation for wild-type and mutant strains obtained from single cells in isolation indicates that the intracellular cAMP dynamics is governed by a PI3kinase-TORC2-actin dependent feedback loop. Collective cAMP oscillations in populations of cells under perfusion reveal a sharp phase transition between a decoupled state and collective behavior for a range of cell densities and dilution rates. These observations together with a mathematical model that we have constructed, suggest that the intact population is able to drive itself to this transition spontaneously during development.

Towards a quantitative input-output relationship in the Dictyostelium cAMP relay response

We revisit the input-output relationship between extracellular cAMP stimulus and intracellular response by live-cell imaging. Single Dictyostelium cells in isolation were stimulated via perfusion over extended periods of time (tens of minutes) with cAMP concentrations that range over 7 orders of magnitude. Three typical response regimes are observed: 1) In the 10pM to 1nM range cells respond stochastically with a single cytosolic cAMP pulse that adapts perfectly on a fast ?3 minute time scale. 2) In the 1nM to 1μM range cells also respond with a fast pulse but cytosolic cAMP continues to oscillate afterwards upon tonic stimulus. These internal oscillations have a damped behavior that is stimulus dependend, but the cells do not fully adapt. 3) In the 1μM-10μM range cytosolic cAMP concentration oscillates continuously but damping is no longer observed. Fourier power spectra for the internal cAMP oscillations reveal periods ranging from 2 to 6 minutes for applied concentrations of 10μM to 1nM, respectively. Damping ranges from minute- (100pM) to hour- (100nM) timescales. The input-output relationship, quantified by computing the concentration integral over the stimulated time interval, shows a sharp transition around 1nM. Furthermore, using a purified version of the sensor protein we estimate both the absolute cytosolic cAMP concentration as well as the number of molecules released by a single cell during a cAMP pulsation.

Modeling adaptation and intrinsic cytosolic cAMP oscillations in isolated Dictyostelium cells

FRET-based measurements of cytosolic cAMP at the single-cell level have enabled us to reexamine the underlying biochemical pathways that give rise to the oscillations and waves of extracellular cAMP. Based on our recent live-cell imaging data from wildtype and mutant strains under various stimulus conditions, we propose a reaction-kinetics model that describes how ligand-binding to the receptor regulates ACA through multiple regulatory modules consisted of Ras, PI3K, TORC2 and F-actin. Our numerical and analytical studies show that a putative negative feedback regulation of F-actin/ACA is necessary to account for the sustained intracellular cAMP oscillations in cells under persistent stimulation. The key signature of the cAMP response that we have experimentally uncovered is the existence of two adaptation kinetics of distinct time-scales. Based on the model simulations, we suggest that the fast activation and adaptation of a cAMP response results from the regulation of PIP3 whereas the slow time scale adaptation derives from actin-dependent inhibition of ACA. By extending the single-cell studies to a multicellular context, we demonstrate that the putative negative feedback and the resulting intrinsic intracellular cAMP oscillations are essential for the onset of the extracellular cAMP oscillations at the population level.

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