Dictyostelium discoideum is commonly referred to as slime mold. It lives in soil and leaf litter while feeding on bacteria. What’s interesting about this organism is that it spends part of its life cycle in a unicellular form and the other part as a multicellular slug or fruiting body. Because of the simplicity of its multicellular form and its ability to shift from unicellularity to multicellularity, researching D. discoideum can bring insight into cell differentiation and multicellularity. A crucial part of researching how D. discoideum is able to form multicellular bodies from its unicellular components is understanding how those single cells communicate with each other in these aggregation and differentiation processes.
Previous knowledge of cell-cell signaling in D. discoideum
Previous studies have identified cAMP as the main signaling molecule in D. discoideum [1,2]. Cells positively chemotax towards cAMP, and this chemotaxis plays a crucial role when cells aggregate to form the multicellular form of the slug [1,2,3]. cAMP has also been shown to play a role in the formation of the fruiting body, by coordinating stalk and spore maturation . As a result of many years of study, elaborate signal-transduction pathways that show this regulation by cAMP have been created [1,2] (Figure 1). These pathways depict cAMP’s role in D. discoideum cell movement/differentiation and all the other major proteins involved  (Figure 1).
Ca2+ chemotaxis in D. discoideum
Recent studies propose that, while cAMP may be the main signaling molecule involved in D. discoideum cell communication, Ca2+ may also play an important role as experiments have shown that cells exhibit chemotaxis towards Ca2+ . Many examples of eukaryotic cells, even human cells, have been shown to respond chemotactically towards Ca2+ . Some of these examples include keratinocytes, osteoblasts, macrophages, mammalian gonadotropin-releasing hormone (GnRH), mouse hematopoietic stem cells, and bracken fern spermatozoids . Concentrations of Ca2+ in media have also been shown to enhance basic cell motility by increased the speed of cellular movement . Ca2+ is even be affected by cAMP levels, as cAMP causes fluctuations in concentrations of extracellular Ca2+ . These factors suggest that D. discoideum amoebas may also show positive chemotaxis towards Ca2+ .
In order to determine the behavior of D. discoideum cells in the presence of molecular gradients, researchers created a microfluidic chamber  (Figure 2). Figure 2 shows a diagram of the customized chamber and a look into how it works. This microfluidic chamber is able to create stable spatial gradients of Ca2+ and cAMP by pumping a buffer from one reservoir pump and a buffer and the chemoattractant of interest from the other reservoir pump. Figure 2D shows the area where the cell behavior is observed. Fluorescence was used for visualizations of chemoattractant gradients.
Behavior of cells in absence of chemoattractant and in presence of cAMP gradient
To make sure the chamber was effective, cell behavior in the absence and presence of a cAMP gradient was observed  (Figure 3). As expected, when there was no chemoattractant gradient, the cells moved on average to the general direction of flow (Figure 3A,B). Chemotaxis is measured by two parameters: the chemotactic index (CI) and the percentage of cells moving up the concentration gradient  (Figure 3C). The CI was calculated by getting the net movement towards the source of chemoattractant divided by the total distance moved . As expected, when in the presence of a cAMP gradient, the cells moved in the direction of the gradient (Figure 3D,E). There was no difference in cell behavior whether the facilitating ion in the buffer was 40 mM K+ or 10 mM Ca2+ (Figure 3). These expected observations showed that the customized microfluidic chamber was an effective research tool for this study.
Behavior of cells in Ca2+ gradient
In order to determine if D. discoideum cells use Ca2+ as a means of communication when aggregating, cell behavior was observed when Ca2+ gradients were generated in the microfluidic chamber  (Figure 4). In both cases of Ca2+ gradients (from top to bottom and from bottom to top), the D. discoideum cells exhibited positive phototaxis as they moved alongside the chemoattractant gradient (Figure 4). This shows that unicellular amobaes could be aggregating into multicellular bodies by means of Ca2+ gradients.
Reversing the gradient during Ca2+ chemotaxis
Researchers also observed cell behavior when the direction of a Ca2+ gradient was changed  (Figure 5). Cells were exposed to a Ca2+ gradient that was pointing upwards for 11 minutes, before reversal was done where the gradient switched to pointing downwards for another 11 minutes (Figure 5). It was found that the cells their locomotion direction to match up with the gradient (Figure 5). This was further evidence that D. discoideum is chemotactic to Ca2+.
Behavior of cells in parallel and opposing gradients of cAMP and Ca2+
To determine the effects of using both cAMP and Ca2+, parallel and opposing gradients of the two chemoattracts were used  (Figure 6). Using this strategy, researchers were able to determine if these two chemical gradients had a stronger effect when present together (parallel gradients) and/or if one chemical had a stronger effect than the other (opposing gradients) . When the cells were placed in parallel chemical gradients, the cells underwent chemotaxis up both gradient with a higher CI than when placed in just a single chemical gradient (Figure 6A,C). This means that the effect of using both cAMP and Ca2+ together is stronger than when either is alone . When the cells were placed in opposing chemical gradients, most cells moved in the direction of the cAMP gradient, with a lower CI than when in cAMP gradient alone (Figure 6B,C). This shows the cAMP is a stronger chemoattractant than Ca2+. The lower CI when cells are in an opposing gradient means that cells are more attracted to cAMP when an opposing Ca2+ gradient is not present.
The study by Scherer et al. 2010 ultimately shows that Dictyostelium discoideum is attracted to Ca2+, and thus Ca2+ could potentially serve as one of the chemoattractants used by unicellular amoebas for aggregation into multicellular bodies. A strength of this study is that the researchers went beyond and conducted a few more mini experiments to determine the level of Ca2+ chemotaxis relative to cAMP chemotaxis. It has been found that cAMP and Ca2+ are more effective were present together, than when either is alone. Also, cells are more attracted to cAMP than Ca2+. Even though Ca2+ could be a potential molecule used in cell aggregation, very steep gradients of Ca2+ are required . A weakness of this study was that they were not able to determine if those steep gradients of Ca2+ occur naturally. Future studies could be done to see how these Ca2+ gradients are developed in the amoeba colonies.
- Kimmel, A., and Parent, C., 2003. The Signal to Move: D. Discoideum Go Orienteering Science 300:1525-1527. http://www.sciencemag.org.www.library.gatech.edu:2048/content/300/5625/1525.full
- Schaap, P., 2011. Evolutionary crossroads in development biology: Dictyostelium discoideum, Development 138:387-396.
- Scherer, A., Kuhl, S., Wessels, D., Lusche, D., Raisley, B., and Soll, D., 2010. Ca2+chemotaxis in Dictyostelium discoideum, Journal of Cell Science 123:3756-3767. http://jcs.biologists.org.www.library.gatech.edu:2048/content/123/21/3756.full