to BioTechniques free email alert service to receive content updates.
Cyclic stretch of the substratum using a shape-memory alloy induces directional migration in Dictyostelium cells
 
Yoshiaki Iwadate1,2 and Shigehiko Yumura1
Full Text (PDF)
Supplementary Material
Supplementary Movie 1. Motion of stretching device using SMA as a power source. (.mov)
To generate the power to stretch the elastic sheet, 4 pieces of coiled SMA were connected in parallel to the left edge of the sheet. The time cycle was 30 s. The movie is shown 5× faster than real time.
Supplementary Movie 2. Dictyostelium cells under the cyclic stretching stimuli. (.mov)
This movie was prepared for demonstration. The densities of cells in this movie were higher than that of the trajectory measurement (see Figure 2 in the main text). The time cycle was 30 s. The movie is shown 10× faster than real time.
Supplementary Movie 3. Migration of Dictyostelium cells under the cyclic stretching stimuli. (.mov)
The images where the substratum shrank in Movie 2 were extracted and accumulated sequentially. The movie is shown 450× faster than real time.

From the 30% stretching, stiffening in the direction of stretching and softening in the perpendicular direction should take place due to the Poisson's effect. However, between the shrunken and 30% stretched sheets without cyclic stretching, there was no significant difference in |sin θ| values [0.64 ± 0.042, SEM (n = 50) and 0.65 ± 0.041 (n = 55)] or migrating speed [6.7 ± 0.038 (n = 50) and 5.8 ± 0.20 (n = 55) µm/min, Figure 2K and 3I, P > 0.01, ANOVA]. These results indicate that the heterogeneous rigidity of the sheet did not induce directional cell migration. Thus, in the following experiments, we regarded the cell migration on the fixed shrunken sheet without cyclic stretching as a control where cells migrated randomly. When the significant difference of |sin θ| (calculated by one-way ANOVA and Ryan's method) between a condition and the control was <0.01, we regarded the migration as directional.

When the cyclic stretching stimuli of 30% stretching ratio and 30 s time cycle were applied (Figure 2, G and J), |sin θ| [0.78 ± 0.031 (n = 55)] was significantly higher than that of the control [0.64 ± 0.042 (n = 50), Figure 3I, P < 0.01]. The average migrating speed of cells with cyclic stretching [8.2 ± 0.31 µm/min (n = 55)] was significantly higher than that of control [6.7 ± 0.38 µm/min (n = 50), Figure 2K, P < 0.01]. These results clearly indicate that the Dictyostelium cells determine their migrating direction and increase their speed in response to the mechanical stimuli from the substratum.

Next, we examined the relationship between the stretching ratio and the degree of the directionality of the migration. Cell migrations under the cyclic stretching stimuli of 10-s time cycle and different stretching ratios (10%, 20%, and 30%) were analyzed (Figure 3, A–F, I). Figure 3, A, C, and E, shows the trajectories of migrating cells under each stretching ratio, respectively. Figure 3, B, D, and F, shows the frequencies of cell migrating direction. The |sin θ| values in each condition are shown in Figure 3I. Under 10% cyclic stretching stimuli, the cells migrated randomly (Figure 3, A and B). There was no significant difference in |sin θ| between the 10% cyclic stretching stimuli [0.64 ± 0.029 (n = 89)] and the control condition [0.64 ± 0.042 (n = 50)]. When 20% cyclic stretching was applied, two small peaks are shown at 90° and 270° in Figure 3D, although there is no significant difference in |sin θ| between the 20% cyclic stretching stimuli [0.69 ± 0.036 (n = 61)] and the control [0.64 ± 0.042 (n = 50), Figure 3I]. Under the 30% cyclic stretching stimuli, the shape of the superimposed trajectories was vertically long (Figure 3E). Two clear peaks are shown at 90° and 270° in Figure 3F. The |sin θ| under the 30% cyclic stretching stimuli [0.80 ± 0.037 (n = 50)] was significantly larger than that of the control [0.64 ± 0.042 (n = 50), Figure 3I, P < 0.01].

To examine the effect of time cycle, cell migrations under cyclic stretching stimuli of 20% stretching ratio and 5-s time cycle (Figure 3, G and H) was compared with that of 20% stretching ratio and 10-s time cycle (Figure 3, C and D). The shape of the superimposed trajectories was vertically long (Figure 3G). The peaks at 90° and 270° in Figure 3H is clearly higher than that in Figure 3D. The value of |sin θ| under cyclic stretching stimuli of 20% stretching ratio and 5-s time cycle [0.77 ± 0.030 (n = 85)] was significantly larger than that of the control [0.64 ± 0.042 (n = 50), Figure 3I, P < 0.01].

On the other hand, the speeds of cell migrations under cyclic stretching stimuli of 10%, 20%, and 30% stretching ratios and a 10-s time cycles, and 20% stretching ratio and 5-s time cycles [10.1 ± 0.25 (n = 89), 9.5 ± 0.35 (n = 61), 8.7 ± 0.34 (n = 50), 10.2 ± 0.12 (n = 85) µm/min, ** in Figure 1J] were significantly higher than that of the control [6.7 ± 0.38 µm/min (n = 50), * in Figure 1J, P < 0.01].

Cells can perform persistent migration even in the absence of an external chemoattractant (23). In order to generate a polarity and migrate in a certain direction without any chemoattractant, cells presumably sense mechanical stimuli from the substratum. For example, fibroblasts sense rigidity of the substratum and move toward rigid areas (the so-called durotaxis) (24,25,26). To ascertain whether the migrating direction of cells is regulated by mechanical inputs from the substratum, detailed measurements of migrating cell trajectories during the application of continuous mechanical stimuli from the substratum are required [as is the case with chemotaxis (27) and galvanotaxis (28,29)]. In this study, the trajectory analysis of migrating cells under continuous mechanical stimuli from the substratum was performed with our new stretching device using SMA. This new device allowed us to demonstrate that mechanical stimuli influence the migration direction of the cells.

  1    2    3    4    5