# Self-organized Vortex State in Two-dimensional Dictyostelium Dynamics

Spontaneous organization of self-propelled particles can be found in a variety of systems. Examples include the flocking of birds , the movement of traffic  and pedestrians  and the collective motion of ants . Not surprisingly, the physics of self-propelled particles has recently attracted considerable attention . The systems have been theoretically analyzed using continuum, Navier-Stokes like, equations and discrete numerical models. The numerical models have in common that the objects are treated as point-particles. However, in a variety of systems the actual shape and plasticity of particles play an important role and hence they can not be modeled in this oversimplified manner. In this Letter we present experimental data in one such system, Dictyostelium discoideum cells, and introduce a model that treats cells as deformable objects rather than point particles. Our results provide evidence for a localized vortex state in this biological system.

The developmental dynamics whereby Dictyostelium discoideum is transformed from a solitary amoeba state to a functional multicellular organism is of interest to both biologists and non-equilibrium physicists . Past efforts have elucidated the nonlinear chemical wave signals used to guide aggregation  as well as the chemotactic instability  which causes a density collapse to one-dimensional “streams” of incoming cells. However, much less is known regarding subsequent events, especially with regard to organized cell motion in the later, multicellular stages of the day-long developmental process.

Dictyostelium cells are grown in liquid media and plated without nutrients onto a glass surface . An additional thin layer of agarose is then overlaid on the cells. This has the effect of restricting cell motion to the plane; in fact, the multicellular states that form are at most a few monolayers deep. Cells aggregate normally and form round ”pancake” structures. In Fig. 1a, we have presented a typical snapshot of the system. In each of the observed structures, the cells have organized their motion into a coordinated vortex state in which they rotate around the center of the pancake. The rotation can be either clockwise or anti-clockwise depending on initial conditions and can persist for tens of hours. Fig 1b shows a close-up of one structure, where now the cells have been illuminated by using a strain in which the gene for green fluorescent protein  has been fused to the CAR1 (cyclic AMP receptor) gene ; the expression of this gene leads to a membrane-localized fusion protein which causes the cell to be fluorescently outlined, as shown. This new protocol for Dictyostelium development allows one to track cell motions in much greater detail than has been possible to date.

It has been previously noted  that coherent rotational motion can often be seen in three dimensional Dictyostelium mounds, albeit as a short-lived transient prior to cell-type sorting and tip formation at the mound apex. This motion has been attributed  to cells moving chemotactically to rotating waves of cyclic AMP. To test this hypothesis, we have repeated our experiments with a non-signaling strain of Dictyostelium . Aside from the need for a higher initial density to overcome the inability of the system to support long-range aggregation, the system behaves in a similar manner and produces rotating vortex structures. Thus, guidance via cAMP waves is not a necessary ingredient for organized rotational motion. Instead, we suspect a self-organization of the system similar to that seen in molecular dynamics simulations of a confined set of particles undergoing dissipative collisions, albeit with deformable particles and without an artificially imposed box.

To verify that such a self-organized state is indeed possible, we turn to a new model of cell motion. Following Glazier and Graner , we assume that cells move via (roughly) volume-preserving fluctuations of their shape. These fluctuations are driven by an effective free energy  which incorporates specific biophysical mechanisms appropriate to Dictyostelium cells. First, cells stick to each other and tend to move in a manner which maximizes cell-cell boundaries over cell-medium ones . In addition, each cell contains an active cytoskeleton which can generate forces by cycles of front protrusions and back retractions . This force appears in our model as a potential which preferentially accepts fluctuations which move the cell in the direction of this propulsive force. To qualitatively verify this methodology, we have simulated the movement of a single self-propelled particle, as shown in Fig. 2. The sequence clearly illustrates the deformability of the cell and reproduces qualitatively the observed movement of a Dictyostelium cell.

In the aforementioned previous work on flocking and related systems, a key insight is that local interactions can cause local velocity correlations which then causes a macroscopically ordered state to emerge. In our model, this suggests that one requires that a cell adjust its own propulsive force based on its interactions with its neighbors. We have investigated several mechanisms whereby this could be incorporated in our model. The most biologically plausible assumption is that the direction of the force is updated so as to better match the forces exerted by neighboring cells - essentially a minimal frustration hypothesis, which is in accord with what one observes directly in the video-microscopy. The results that follow were derived for this specific assumption, but depend very weakly on any details beyond the basic correlation idea.

To investigate the possibility of coherent vortex motion we started simulations with 100 square cells stacked in a square surrounded by medium. The force direction of each cell was chosen at random. A snapshot of a typical final state is shown in Fig 3 which shows the boundaries of the cells and the force direction as a line which starts at the center-of-mass (CM) of the cell and which points in the direction of the force. The cells form a roughly circular patch and are rotating around the center of the patch. The final state is typically reached after a transient of 100-1000 Monte Carlo steps (MCS) . Depending on the initial conditions, the cells will rotate either clockwise or anti-clockwise. Other than the sense of rotation and the duration of the transient phase, nothing depends on the starting state. Similar results were obtained for different numbers of cells. The existence of a localized rotating state is a robust consequence of the model, present as long as the cells are sufficiently cohesive ($`J_{cm}>J_{cc}`$). Hence, we have proven that simple models based directly on the observed cell motions can indeed account for the self-localized vortex.

To further test our model, we consider the angular velocity of cells as a function of radius. In the experiments, the angular velocity was obtained by direct tracking of cells. This tracking is greatly facilitated by the use of the cell-membrane outlining approach as shown in Fig. 1b. In detail, we have taken six separate sequences of 15 min. each and measured the angular velocity every 8 s. During each sequence the radius of the individual cells changed little. Next, we grouped the data in radius intervals of 4 $`\mu m`$ and calculated the average velocity and the standard deviation for each interval. The data is shown in Fig. 4 where the vertical bars represent one standard deviation. The overall time scale corresponding to a MCS was adjusted to provide the best fit of the model (solid line) to the data; this yields 1 $`MCS`$ as $`0.006min`$. As a consistency check, we note that this gives an isolated cell velocity of 8 $`\mu `$m/min, which is very close to the experimentally reported value of 10 $`\mu `$m/min . Although there is some way to go for a fully quantitative theory, our simple model does surprisingly well in capturing both the tendency of the cell-cell interaction to speed up the motion and the tendency of cells to slip as they move past each other, thereby limiting the angular velocity at large radii.

What additional predictions does our model make? As the model proposes that cell-cell adhesion is the cause of the localized coherent state, mutant strains with reduced adhesion should not be able to form this structure. Also, mutants with weakened cytoskeletons would not be able to organize their motion. As chemical signaling is not necessary, disrupting the external cAMP concentration should have minimal effects. Finally, our model suggests that the time scale for organization should roughly be tens of minutes for aggregates with hundreds of cells; this is in qualitative agreement with our observations (data not shown).

Vortex structures have been seen in other microorganism systems, namely the nutrient-limited spreading of the newly named bacterium Paenibacillus dendritiformis , and in Bacillus circulans . The motion of bacteria occurs through flagella and is thus fundamentally different from the amoeboid motion of Dictyostelium. However, it is tempting to speculate that in these cases as well velocity correlations induced by cell-cell interactions as well as cohesive forces may be enough to account for these structures . The fact that these disparate systems exhibit such strikingly similar non-equilibrium structures offers comfort to the physicist proposing a simplified model for an inscrutably complex biological process.

In summary, we have documented the existence of a localized, coherent vortex state in Dictyostelium. Furthermore, we have argued both experimentally and via construction of a new model that the coherent motion is self-organized and not the result of a rotating chemical-wave guiding the motion. The advantage of this deformable-cell model is that it allows for the direct comparison of simulation with observation. In the future, we plan to extend our calculations to the later stage processes of cell sorting and slug formation.

We would like to thank A. Kuspa and P. Devreotes for providing some of the Dictyostelium strains used in the experiments. Also, one of us (HL) acknowledges useful conversations with E. Ben-Jacob. This work was supported in part by NSF DBI-95-12809.