Figure 7.

WASP KO cells are defective in their ability to interpret the topological features of their environment. (A) WASP puncta enrich at nanoridges as cells migrate over patterns. Scale bar is 10 μm. (B) Maximum intensity projection of data in A shows that WASP localizes to nanoridge peaks. An intensity profile for WASP and the pattern along the vertical line in the merged image is shown below. Scale bar is 5 μm and 1 μm in the inset. (C) There is a modest difference in the total distance traveled by WT (purple) and WASP KO (guide 1 [g1] = blue, g2 = magenta) cells plated on flat substrates. n_WT = 253 cells, n_KO1 = 215 cells, and n_KO2 = 181 cells collected across three experiments. Total distance traveled: WT = 246 ± 1 μm, WASP KO1 = 210 ± 6 μm, and WASP KO2 = 201 ± 12 μm on the replicate level. Replicate means are denoted throughout the figure by enlarged markers. Only one of the WASP KO clones was significantly different from WT. For this case, P = 0.026 by a paired two-tailed t test on replicate means. In the other case, P = 0.13. See also Video 7. (D) The total distance traveled by WASP KO cells is significantly less than that of WT cells on nanoridged substrates due to defective movement along the patterns (here, the x axis). n_WT = 188 cells, n_KO1 = 172 cells, and n_KO2 = 242 cells collected across three experiments. Total distance traveled: WT = 273 ± 17 μm, WASP KO1 = 194 ± 8 μm, and WASP KO2 = 202 ± 5 μm on the replicate level. Total distance traveled in x: WT = 241 ± 19 μm, WASP KO1 = 139 ± 8 μm, and WASP KO2 = 149 ± 6 μm on the replicate level. Total distance traveled in y: WT = 78 ± 1 μm, WASP KO1 = 102 ± 6 μm, and WASP KO2 = 102 ± 5 μm on the replicate level. *, P < 0.05, which was determined by a paired two-tailed t test on replicate means. Unless labeled, the difference is not significant. P_{total, WT/KO1} = 0.018, P_{total, WT/KO2} = 0.028, P_{x, WT/KO1} = 0.023, P_{x, WT/KO2} = 0.023, P_{y, WT/KO1} = 0.061, P_{y, WT/KO2} = 0.035. See also Video 8. (E) Ratio of aligned to perpendicular migration reported in D highlights defective nanopattern sensing by WASP KO cell lines. Average ratio of total aligned to total perpendicular distance traveled: WT = 4.02 ± 0.23, WASP KO1 = 1.47 ± 0.17, and WASP KO2 = 1.57 ± 0.15 on the replicate level. *, P < 0.05, which was determined by a paired two-tailed t test on replicate means. P_WT/KO = 0.012 and P_WT/KO2 = 0.0078. (F) Bright field images reveal clear alignment of WT cells to horizontal nanoridges, while WASP KO cells largely failed to align. Scale bar is 5 μm. (G) WT and WASP KO cell trajectories over 1 h for cells migrating on flat substrates (top) and nanoridged substrates (bottom). Image dimensions are ∼665 × 665 μm. (H) The MSDs of WT and WASP KO cells are indistinguishable on flat substrates (top), while the MSDs of WASP KO cells are significantly less than that of WT cells on nanoridged substrates (bottom). Shading denotes a 95% confidence interval. Trajectories are the same as those assayed in C and D, respectively. Of note, the MSD of WT cells is 10-fold greater on nanoridged substrates than on flat substrates. (I) The persistence ratios of WT and WASP KO cells are indistinguishable on flat substrates (top), while the persistence ratios of WASP KO cells are significantly less than that of WT cells on nanoridged substrates (bottom). Shading denotes a 95% confidence interval. Trajectories are the same as those assayed in C and D, respectively. (J) Model of the role of WASP and WASP-mediated actin polymerization in topology sensing and contact guidance.