Figure 3.
MT depletion causes migratory failure due to hyperactive and destabilized actomyosin contractility. (a) Lifeact-GFP–expressing DC migrating within a path choice device. Scale bar, 10 µm. (b) Nocodazole-treated Lifeact-GFP–expressing cell migrating within a path choice device. Note that the cell extends elongated protrusions into different channels. Red arrowhead denotes a cell rupturing event. See also Video 5. (c) Frequency of cell rupturing events during migration within path choice devices of n = 43 cells (control) and n = 44 cells (nocodazole; Noco.) ±SD of N = 2 experiments. (d) Time-lapse sequence of a cell migrating within a linear microchannel. See also Video 6. Scale bar, 10 μm. (e) Nocodazole-treated cell migrating in the same configuration as in d.Scale bar, 10 μm. (f) Cell treated with a combination of Y27632 plus nocodazole migrating as shown in d. Scale bar, 10 µm. (g) Migration speed of control, nocodazole-treated, or double-treated cells using Y27632 and nocodazole within microchannels (n = minimum of 74 cells per condition from N = 4 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of one-way ANOVA with Tukey’s test; *, P ≤ 0.05; ****, P ≤ 0.0001. (h) Persistence of control, nocodazole-treated, or double-treated cells using Y27632 and nocodazole within microchannels (n = minimum of 74 cells per condition from N = 4 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of Kruskal-Wallis with Dunn’s test; ***, P ≤ 0.001; ****, P ≤ 0.0001. (i) DCs migrating within a collagen gel either non-treated (control) or double-treated with Y27632 and nocodazole. Note the different time intervals per condition. Red arrowheads indicate the loss of cellular coherence in the double-treated cell. Scale bar, 10 µm. See also Video 4. (j) Automated analysis of the y-directed speed of non-treated (NT), nocodazole-treated, or double-treated cells using Y27632 and nocodazole. Plot shows mean population migration velocities over time ± SD from N = 4 experiments. (k) Lifeact-GFP–expressing DC double-treated with Y27632 plus nocodazole migrating as in panel a. Red arrowhead denotes cell rupturing and loss of cellular coherence. Scale bar, 10 µm. (l) Frequency of cell rupturing events during migration within the path choice device of n = 40 cells (control) and n = 80 cells (Y./N.) ±SD of N = 2 experiments. (m) Lifeact-GFP–expressing DC treated with Y27632 migrating as in panel a. Note the extended protrusions are reaching far into separate channels without generating a productive decision within the indicated time. Scale bar, 10 µm. Noco., nocodazole; n.s., not significant; Y./N., double-treated with Y27632 and nocodazole.

MT depletion causes migratory failure due to hyperactive and destabilized actomyosin contractility. (a) Lifeact-GFP–expressing DC migrating within a path choice device. Scale bar, 10 µm. (b) Nocodazole-treated Lifeact-GFP–expressing cell migrating within a path choice device. Note that the cell extends elongated protrusions into different channels. Red arrowhead denotes a cell rupturing event. See also Video 5. (c) Frequency of cell rupturing events during migration within path choice devices of n = 43 cells (control) and n = 44 cells (nocodazole; Noco.) ±SD of N = 2 experiments. (d) Time-lapse sequence of a cell migrating within a linear microchannel. See also Video 6. Scale bar, 10 μm. (e) Nocodazole-treated cell migrating in the same configuration as in d.Scale bar, 10 μm. (f) Cell treated with a combination of Y27632 plus nocodazole migrating as shown in d. Scale bar, 10 µm. (g) Migration speed of control, nocodazole-treated, or double-treated cells using Y27632 and nocodazole within microchannels (n = minimum of 74 cells per condition from N = 4 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of one-way ANOVA with Tukey’s test; *, P ≤ 0.05; ****, P ≤ 0.0001. (h) Persistence of control, nocodazole-treated, or double-treated cells using Y27632 and nocodazole within microchannels (n = minimum of 74 cells per condition from N = 4 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of Kruskal-Wallis with Dunn’s test; ***, P ≤ 0.001; ****, P ≤ 0.0001. (i) DCs migrating within a collagen gel either non-treated (control) or double-treated with Y27632 and nocodazole. Note the different time intervals per condition. Red arrowheads indicate the loss of cellular coherence in the double-treated cell. Scale bar, 10 µm. See also Video 4. (j) Automated analysis of the y-directed speed of non-treated (NT), nocodazole-treated, or double-treated cells using Y27632 and nocodazole. Plot shows mean population migration velocities over time ± SD from N = 4 experiments. (k) Lifeact-GFP–expressing DC double-treated with Y27632 plus nocodazole migrating as in panel a. Red arrowhead denotes cell rupturing and loss of cellular coherence. Scale bar, 10 µm. (l) Frequency of cell rupturing events during migration within the path choice device of n = 40 cells (control) and n = 80 cells (Y./N.) ±SD of N = 2 experiments. (m) Lifeact-GFP–expressing DC treated with Y27632 migrating as in panel a. Note the extended protrusions are reaching far into separate channels without generating a productive decision within the indicated time. Scale bar, 10 µm. Noco., nocodazole; n.s., not significant; Y./N., double-treated with Y27632 and nocodazole.

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