Cell dispersal by localized degradation of a chemoattractant

Abstract

Chemotaxis, the guided motion of cells by chemical gradients, plays a crucial role in many biological processes. In the social amoeba Dictyostelium discoideum, chemotaxis is critical for the formation of cell aggregates during starvation. The cells in these aggregates generate a pulse of the chemoattractant, cyclic adenosine 3',5'-monophosphate (cAMP), every 6 min to 10 min, resulting in surrounding cells moving toward the aggregate. In addition to periodic pulses of cAMP, the cells also secrete phosphodiesterase (PDE), which degrades cAMP and prevents the accumulation of the chemoattractant. Here we show that small aggregates of Dictyostelium can disperse, with cells moving away from instead of toward the aggregate. This surprising behavior often exhibited oscillatory cycles of motion toward and away from the aggregate. Furthermore, the onset of outward cell motion was associated with a doubling of the cAMP signaling period. Computational modeling suggests that this dispersal arises from a competition between secreted cAMP and PDE, creating a cAMP gradient that is directed away from the aggregate, resulting in outward cell motion. The model was able to predict the effect of PDE inhibition as well as global addition of exogenous PDE, and these predictions were subsequently verified in experiments. These results suggest that localized degradation of a chemoattractant is a mechanism for morphogenesis.

Keywords: Dictyostelium discoideum; chemotaxis; dispersal; repulsion.

Fig. 1.

(A) Micrographs showing cell motion, visualized using dense optical flow, near a dispersing aggregate at four different times. Dispersal started at $t=0$, and inward/outward motion is indicated in red/blue, while the aggregate center is marked by the yellow x. (Scale bar: 50 $\mathrm{\mu }$m.) (B) Examples of single-cell trajectories during outward (indicated by black arrow) and inward motion (indicated by blue arrow), taken from the second and third panels (black and blue boxes) in A. (Scale bar: $50\hspace{0.17em}\mathrm{\mu }\text{m}$.) The symbols indicate the cell location at each frame (separated by 1 min). (C) Spatially averaged inward/outward motion (red/blue), normalized by total cell area, for cells within a distance of 100 $\mathrm{\mu }$m and 300 $\mathrm{\mu }$m from the aggregate center as a function of time. (D) Median CI for all cells within the same region as in B. (E) Histogram of the median aggregate size for dispersing and oscillating aggregates and for nondispersing aggregates. ***$P<0.001$.

Fig. 2.

(A) Micrographs of a dispersing aggregate of cells expressing Flamindo2 and the corresponding fluorescent intensity snapshots. (Scale bar: 50 $\mathrm{\mu }$m.) (B) Inward/outward optical flow (red/blue) averaged over the ranges 60 $\mathrm{\mu }$m to 120 $\mathrm{\mu }$m. (C) Mean fluorescent intensity of Flamindo2 cells, averaged over 75 $\mathrm{\mu }$m to 150 $\mathrm{\mu }$m from the aggregate center (green line) and within $20\hspace{0.17em}\mathrm{\mu }$m from this center (black line), as a function of time. (D) As in C, but now for t = 45 min to 60 min after the onset of dispersal. (E) Cross-correlation of the green and black signal in D, demonstrating that the change in intensity in the aggregate occurs $\sim$30 s before the change in cells away from the aggregate. (F) Cross-correlation of the inward motion and the fluorescent intensity sampled 50 min to 20 min before the onset of dispersal (Top) and 40 min to 70 min after the onset of dispersal (Bottom). (G) Period of cAMP signaling just before and right after the onset of dispersal (${\mathrm{N}}_{agg}$ = 14). **$P<0.01$.

Fig. 3.

(A) Schematic drawing of the setup of the 2D computer simulations. A circular aggregate of fixed size (50-$\mathrm{\mu }$m radius) periodically secretes cAMP signals as well as a constant amount of PDE. The computational domain had a radius of 1,000 $\mathrm{\mu }$m, and the cAMP concentration at its boundary was assumed to be constant. (B) Phase diagram for $cAM{P}_{back}=12$ nM, indicating parameter values for which PDE degradation resulted in dispersal. (C–F) Radial concentration gradients and corresponding profiles (Inset) plotted for $L_{PDE}=100\hspace{0.17em}\mathrm{\mu }\text{m}$ and $k_{PDE}=0.6\hspace{0.17em}{\text{s}}^{-1}$ (C), $k_{PDE}=0.04\hspace{0.17em}{\text{s}}^{-1}$ (F), and $k_{PDE}=0.004\hspace{0.17em}{\text{s}}^{-1}$ (D), and for $L_{PDE}=20\hspace{0.17em}\mathrm{\mu }\text{m}$ and $k_{PDE}=0.04\hspace{0.17em}{\text{s}}^{-1}$ (E). The minimum gradient for direction sensing is indicated by the black line.

Fig. 4.

(A) Net cell motion during a cAMP cycle (T = 10 s) in response to the global addition of a PDE inhibitor (Left) and of PDE (Right). (B) Radial cAMP concentration profiles and outward cAMP gradient (Inset) are shown at 1 min before (Left) and 6 min after the simulated addition of the inhibitor (Middle) and PDE (Right).

Fig. 5.

(A) Micrograph of a previously nondispersing aggregate after exposure to medium transferred from an aggregate that was dispersing. Motion is visualized using dense optical flow, with blue/red corresponding to motion away from/toward the transferred fluid (yellow “x”). (Scale bar: $50\hspace{0.17em}\mathrm{\mu }$m.) (B and C) Micrographs 7 min before (B) and 7 min after (C) the addition of DTT at $t=0$. Cell motion was calculated by dense optical flow inward/outward from the aggregate (yellow “x”). (Scale bar: $50\hspace{0.17em}\mathrm{\mu }$m.) (D) Average inward/outward cell motion (red/blue) in response to DTT introduced at $t=0$. (E) Mean Flamindo2 fluorescent intensity of the aggregate versus time in response to DTT introduced at $t=0$.

Fig. 6.

(A and B) Micrographs of an initially dispersing aggregate t = −14 min before the addition of PDE1 (A) and t = +14 min after the addition of PDE1 (B). (Scale bar: $50\hspace{0.17em}\mathrm{\mu }$m.) (C) Inward/outward (red/blue) optical flow averaged for cells 80 μm to 160 μm away from the aggregate center. PDE1 was added at t = 0. (D) Total Flamindo2 fluorescent intensity, computed using a 5-min moving average, for cells inside the aggregate (black) and cells 80 μm to 160 μm away from the aggregate center (green).