Sensing of substratum rigidity and directional migration by fast-crawling cells

Living cells sense the mechanical properties of their surrounding environment and respond accordingly. Crawling cells detect the rigidity of their substratum and migrate in certain directions. They can be classified into two categories: slow-moving and fast-moving cell types. Slow-moving cell types, such as fibroblasts, smooth muscle cells, mesenchymal stem cells, etc., move toward rigid areas on the substratum in response to a rigidity gradient. However, there is not much information on rigidity sensing in fast-moving cell types whose size is ∼10 $\mu \mathrm{m}$ and migration velocity is ∼10 $\mu \mathrm{m}/\min$. In this study, we used both isotropic substrata with different rigidities and an anisotropic substratum that is rigid on the $x$ axis but soft on the $y$ axis to demonstrate rigidity sensing by fast-moving Dictyostelium cells and neutrophil-like differentiated HL-60 cells. Dictyostelium cells exerted larger traction forces on a more rigid isotropic substratum. Dictyostelium cells and HL-60 cells migrated in the “soft” direction on the anisotropic substratum, although myosin II–null Dictyostelium cells migrated in random directions, indicating that rigidity sensing of fast-moving cell types differs from that of slow types and is induced by a myosin II–related process.

Figure 1

Traction forces exerted by wild-type and E476K Dictyostelium cells on substrata with different Young's moduli. (a–c) Wild-type cells. (d–f) E476K cells. Young's moduli: 101 Pa (a,d), 281 (b,e), and 463 Pa (c,f), respectively. (a–f) are typical of 20, 27, 24, 20, 21, and 25 cells, respectively. (g,h) Mean and maximum traction forces exerted by wild-type cells on each substratum. (i,j) Mean and maximum traction forces exerted by E476K cells on each substratum. (k,l) Migration velocities of wild-type and E476K cells on each substratum [(k) $n=30$ (101 Pa), 31 (281 Pa), and 29 (463 Pa); (l) $n=20$ (101 Pa), 21 (281 Pa), and 25 (463 Pa)]. Bars: SEM. The $p$ values were calculated using Student's $t$-test (g–j) and one-way ANOVA (k,l). ${}^{*}p<0.01$.

Figure 2

Anisotropic substratum. (a) Schematic view of the substratum. The two-layered PDMS sheet (see Materials and Methods for details) was stretched such that its length doubled. (b) Flexible glass microneedle for evaluating the compliances of the substratum in the $x$ direction and the $y$ direction in (a). (c) Evaluation method. The substratum was pulled by moving the needle (arrow) whose tip adhered to the surface of the substratum. D: deflection of the needle; L: displacement of the tip of the needle. (d,e) Apparent spring constants of the substratum after [(d) $n=14$ ($x$) and 12 ($y$)] and before [(e) $n=12$ ($x$) and 11 ($y$)] the stretching of the sheet. (f,g) A scanning electron microscope image of the surface of the substratum. The rectangle in (f) is enlarged in (g). (h–j) Comparison of adhesion strength in the $x$ direction and the $y$ direction. Single Dictyostelium cells were sacked horizontally with a pipet [(h) schematic and (i) top view image] and pulled by moving it [arrow in (h)]. Adhesion strengths in both directions were estimated from negative pressure at which the cell was detached from the substratum [(j) $n=51$ ($x$) and 48 ($y$)]. Bars: SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.

Figure 3

Crawling migration of wild-type Dictyostelium cells on the anisotropic substratum. (a) Trajectories of migrating cells on the anisotropic substratum, soft in the longitudinal direction (90°–270°) and rigid in the horizontal (0°–180°) direction ($n=114$ from six experiments). (b) Frequencies of directions of migration (θ) calculated from (a). The θ [inset in (a)] is defined as the angle between a horizontal line and the straight line connecting the starting and ending point of each migrating cell during a single experiment (30 min). (c) Trajectories of migrating cells on the control substratum ($n=107$ from ten experiments). (d) Frequencies of directions of migration (θ) calculated from (c). (e) Comparison of DPM values. The DPM values on the anisotropic substratum were significantly higher than those on the control substratum. Dashed horizontal line: 0.64, the value when cells migrate equally in all directions. Bars: SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.

Figure 4

Crawling migration of HL-60 cells on the anisotropic substratum. (a) Trajectories of migrating cells on the anisotropic substratum, soft in the longitudinal direction (90°–270°) and rigid in the horizontal (0°–180°) direction ($n=102$ from five experiments). (b) Frequencies of direction of migration (θ) calculated from (a). (c) Trajectories of migrating cells on the control substratum ($n=116$ from ten experiments). (d) Frequency of direction of migration (θ) calculated from (c). (e) Comparison of DPM values. The DPM values on the anisotropic substratum were significantly higher than those on the control substratum. Dashed horizontal line: 0.64, the value when cells migrate equally in all directions. Bars: SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.

Figure 5

Migration property of fast-moving cell types on the anisotropic substratum. (a) Analysis methods (see Materials and Methods for details). Thick arrow: a vector from the initial location of a cell ($t=T$) to that in the time frame ($t=T+30\mathrm{s}$). The angle (ϕ) between the $x$ axis ($x$) and the vector is regarded as the direction of migration at each time point. Rigid direction: between 315° (–45°) and 45° or between 135° and 225° (gray region); soft direction: between 45° and 135° or between 225° and 315° (white region). (b,c) Migration velocities of Dictyostelium cells on the anisotropic (b) and control substratum (c). (d,e) Probabilities of switch in direction of migration of Dictyostelium cells on the anisotropic (d) and control substratum (e). (f,g) Migration velocities of HL-60 cells on the anisotropic (f) and control substratum (g). (h,i) Probabilities of switch in direction of migration of HL-60 cells on the anisotropic (h) and control substratum (i). The data in (b,d), (c,e), (f,h), and (g,i) were calculated from the migrations seen in Figs. 3 and 3, and Figs. 4 and 4, respectively. Bars: SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.

Figure 6

Crawling migration of $mhc{A}^{–}$Dictyostelium cells on the anisotropic substratum. (a) Trajectories of migrating cells on the anisotropic substratum, soft in the longitudinal direction (90°–270°) and rigid in the horizontal (0°–180°) direction ($n=157$ from six experiments). (b) Frequencies of migrating direction (θ) calculated from (a). (c) Trajectories of migrating cells on the control substratum ($n=163$ from six experiments). (d) Frequencies of migrating direction (θ) calculated from (c). (e) Comparison of DPM values. There was no significant difference in DPM values between the anisotropic and control sheets. Dashed horizontal line: 0.64, the value when cells migrate equally in all directions. Bars: SEM. The $p$ values were calculated using Student's $t$-test.

Figure 7

Crawling migration of $mhc{A}^{–}$Dictyostelium cells expressing myosin II variants on the anisotropic substratum. (a–c) Frequencies of the migrating direction (θ) of 3×ALA cells [(a) $n=138$ from five experiments], 3×ASP cells [(b) $n=128$ from six experiments], and E476K cells [(c) $n=101$ from five experiments] on the anisotropic substratum. The arrows in (a,c) indicate two peaks at 90° and 270°. (d–f) Frequency of direction of migration (θ) of 3×ALA cells [(d) $n=141$ from six experiments], 3×ASP cells [(e) $n=160$ from six experiments], and E476K cells [(f) $n=130$ from five experiments] on the control substratum. (g–i) Comparison of DPM values. The values in the left and right columns in each graph were calculated from the migrations on the anisotropic substratum (a–c) and control substratum (d–f), respectively. In the 3×ALA and E476K cells, the DPM values on the anisotropic substratum were significantly higher than those on the control substratum. Dashed horizontal lines in (g–i): 0.64, the value when cells migrate equally in all directions. Bars in (g–i): SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.

Figure 8

Migration property of 3×ALA and E476K Dictyostelium cells on the anisotropic substratum. (a,b) Migration velocities of 3×ALA cells on the anisotropic (a) and control substratum (b). (c,d) Probabilities of switch in direction of migration of 3×ALA cells on the anisotropic (c) and control substratum (d). (e,f) Migration velocities of E476K cells on the anisotropic (e) and control substratum (f). (g,h) Probabilities of a switch in direction of migration of E476K cells on the anisotropic (g) and control substratum (h). The data in (a,c), (b,d), (e,g), and (f,h) were calculated from the migrations shown in Figs. 7, 7, 7, and 7, respectively. Bars: SEM. The $p$ values were calculated using Student's $t$-test. ${}^{*}p<0.01$.