How do the cells communicate with each other to aggregate into the multicellular forms?


Fruiting body of D. discoideum (http://wever.wordpress.com)

Dictyostelium discoideum

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.

Video of Dictyostelium discoideum aggregation into slug and fruiting body

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 [2]. 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 [1] (Figure 1).

Figure 1. Signal-transduction pathway for cAMP regulation of cell aggregation (Figure 1. Kimmel and Parent 2003)

Ca2+ chemotaxis in D. discoideum

Introduction
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+ [3]. Many examples of eukaryotic cells, even human cells, have been shown to respond chemotactically towards Ca2+ [3]. Some of these examples include keratinocytes, osteoblasts, macrophages, mammalian gonadotropin-releasing hormone (GnRH), mouse hematopoietic stem cells, and bracken fern spermatozoids [3]. Concentrations of Ca2+ in media have also been shown to enhance basic cell motility by increased the speed of cellular movement [3]. Ca2+ is even be affected by cAMP levels, as cAMP causes fluctuations in concentrations of extracellular Ca2+ [3]. These factors suggest that D. discoideum amoebas may also show positive chemotaxis towards Ca2+ [3].

Microfluidic chamber

In order to determine the behavior of D. discoideum cells in the presence of molecular gradients, researchers created a microfluidic chamber [3] (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.

Figure 2. Customized microfluidic chamber manufactured by Translume from optically clear, fused silica according to our specifications. (A) Diagram of microfluidic chamber. The channel is a square tube with sides of 300 µm and a length of 28 mm, shown in blue. Two programmable pumps control the flow rates from reservoirs a and b. (B) The interface of solutions a and b can be visualized by excitation of fluorescein (released by reservoir a) at the intersection of the inlet ports with the channel. (C) As solutions a and b flow through the chamber, they encounter a series of chevron micromixers. The gradient is generated perpendicular to the direction of flow. (D) The area of observation in the channel is adjacent to the cell injection port; the latter is where the cell suspension is introduced into the channel. Efflux occurs through tubing connected to the outlet port. (E–H) Gradient steepness is controlled by the flow rate of the pumps and plotted at different flow rates from data obtained when reservoir a contained fluorescein. The steepness of the concentration gradient of this fluorophore across the width of the chamber was determined using 2D-DIAS software (Soll, 1995) and provided in each plot in nM/µm. (I) The stability of the gradient was demonstrated by measuring fluorescence in images of the channel at 1, 10 and 20 minutes. (Figure 1. Scherer et al. 2010)

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 [3] (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 [3] (Figure 3C). The CI was calculated by getting the net movement towards the source of chemoattractant divided by the total distance moved [3]. 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.

Figure 3. Behaviors of cells migrating in the microfluidic chamber in the absence of a chemoattractant gradient or in cAMP gradients verify the effectiveness of the chamber. The pumps are programmed to deliver at the optimum flow rate for Ca2+. Buffer solutions in both reservoirs contained either 40 mM K+ or 10 mM Ca2+ as the facilitating ion in both the absence (A–C) and presence (D–F) of a cAMP gradient. The ionic content of solutions in flow a and flow b are presented at the top and bottom, respectively, of A,B,D,E, with the direction of flow indicated by the bold arrows pointing to the right. The direction of the cAMP gradient (large, shaded arrows in D and E) is perpendicular to the direction of flow. Stacked perimeter plots of representative cells generated at 8 second intervals by DIAS software are presented in A,B,D,E. Thin arrows alongside each cell indicate the direction of travel. The final outline in each series of perimeter plots is colored blue. Motility parameters for the conditions in A and B are provided in C. Motility parameters for the conditions in D and E are provided in F. No. expts., the number of experiments; No. cells, the number of cells analyzed; +B.I., positive bias index; Inst. vel., instantaneous velocity; Direct. persist, directional persistence (see Results for definitions of parameters). (Figure 2. Scherer et al. 2010)

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 [3] (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.

Figure 4. Cells undergo chemotaxis in a Ca2+ gradient. (A) Perimeter tracks of representative cells undergoing chemotaxis in a gradient of Ca2+ increasing from bottom to top. (B) Perimeter tracks of representative cells undergoing chemotaxis in a gradient of Ca2+ increasing from top to bottom. (C) Raw images of cells, viewed through brightfield microscopy, undergoing chemotaxis up a Ca2+ gradient. (D) Motility and chemotaxis parameters of cells undergoing chemotaxis in a Ca2+ gradient. The direction of the gradients was perpendicular to the direction of flow. A gradient of Ca2+ (A) was generated by filling reservoir a with 10 mM Ca2+ in TB, and reservoir b with TB alone. The contents of the reservoirs are reversed in B. The pumps are programmed to deliver at the optimum flow rate for Ca2+. The large shaded arrows in A–C indicate the directions of the Ca2+ gradients. See Fig. 2 for additional details. (E) Dose–response curve for Ca2+ chemotaxis in which the CI was used to measure the response. Small dots on the x-axis indicate that the concentration approaches 0 as it tends to infinity. (Figure 3. Scherer et al. 2010)

Reversing the gradient during Ca2+ chemotaxis

Researchers also observed cell behavior when the direction of a Ca2+ gradient was changed [3] (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+.

Figure 5. Cells undergoing chemotaxis in a spatial gradient of Ca2+ change direction upon gradient reversal through lateral pseudopod extension. (A,B) The behavior of representative cells upon gradient reversal at 11 minutes (vertical dashed line). The average CI is presented for each cell following the initial and reversed gradient. (C) Difference pictures of the cell in A before and after reversal. Green areas are expansion zones and red areas are contraction zones. Arrows denote direction of translocation. Green arrows denote direction of gradients. (Figure 4. Scherer et al. 2010)

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 [3] (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) [3]. 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 [3]. 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.

Figure 6. Behavior of cells in parallel and opposing cAMP and Ca2+ gradients. (A) Perimeter tracks of representative cells in parallel cAMP and Ca2+ gradients indicate that the Ca2+ gradient enhances chemotaxis up the cAMP gradient. (B) Perimeter tracks of representative cells in opposing cAMP and Ca2+ gradients indicate that the cAMP gradient elicits a stronger chemotactic response than the Ca2+ gradient. (C) Motility and chemotaxis parameters of cells in parallel and opposing gradients. See Fig. 2 for definitions of abbreviated headings. (Figure 5. Scherer et al. 2010)

Conclusions

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 [3]. 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.

References

  1. 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
  2. Schaap, P., 2011. Evolutionary crossroads in development biology: Dictyostelium discoideum, Development 138:387-396.
  3. 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

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