![Lfc−/− DCs exhibit reduced contractile responses. (a) Left: Cell outlines of Lfc+/+ (left) and Lfc−/− (right) DCs migrating within a collagen network along a soluble CCL19 gradient. Scale bar, 10 µm. Right: Graph shows the lengths of cells migrating within a collagen network of n = 85 individual 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 unpaired Student’s t test; ***, P ≤ 0.001. (b) Levels of active RhoA of Lfc+/+ and Lfc−/− cells were determined by luminometry showing the mean intensities ± SD from N = 3 experiments. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. (c) Levels of MLC phosphorylation (pMLC) in Lfc+/+ and Lfc−/− DCs assessed by Western blot analysis. Cells were treated with the indicated compounds (DMSO, CCL21, nocodazole [Noco.], or Y27632 together with nocodazole [Y/N]). (d) Mean fluorescence intensity of phospho-MLC was normalized to GAPDH signal and shown as fold increase relative to DMSO control ± SD. Blots are representative of N = 3 experiments. Annotation above columns indicates results of two-way ANOVA; *, P ≤ 0.1. r.u., relative units. (e) Centrosome localization relative to the nucleus in Lfc−/− DCs migrating under agarose assessed by α- and γ-tubulin costaining (n = 117 cells from N = 2 experiments ±SD). (f) Centrosome position relative to the nucleus of Lfc−/− DCs migrating within a path choice assay. Shown are mean frequencies of n = 49 cells from N = 2 experiments ±SD. cho., choice. (g) MT nucleation from centrosomal origin as determined by α- and γ-tubulin costaining. Blue line indicates the position of the nucleus. Scale bars, 10 µm. (h) Intensity line profiles across the highest γ-tubulin (tub.) signal along the left-right axis (dashed line in g). The purple line indicates γ-tubulin signal intensity. The black line indicates α-tubulin signal distribution. (i) Path choice preference of Lfc+/+ and Lfc−/− DCs migrating within a path choice assay. Shown are mean frequencies of Lfc−/− (n = 49 cells from N = 2 experiments) and Lfc+/+ (n = 79 cells from N = 3 experiments) DCs ±SD. (j) Frequency of cell rupturing events of Lfc+/+ (n = 73 cells from N = 3 experiments) and Lfc−/− (n = 128 cells from N = 3 experiments) DCs while migrating within single constriction–containing microchannels ±SD. (k) Migration speed of nocodazole-treated cells within straight microchannels of n = minimum of 80 cells per condition from N = 5 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. (l) Migratory persistence of nocodazole-treated Lfc+/+ and Lfc−/− DCs within straight microchannels of n = minimum of 80 cells per condition from N = 5 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 ≤ 0.01. n.s., not significant.](https://cdn.rupress.org/rup/content_public/journal/jcb/219/6/10.1083_jcb.201907154/6/jcb_201907154_figs5.png?Expires=1771026860&Signature=Hn7do6SnYTop7ybXbFXRERyMrrBDGARdu4teAILZwqH7LyRLbTvwSJKlJpQvUBY5wkcGocSAxqxmfJc1xtyWkIQRixQRxPo-rZxhKBE-NdRBkFUvDW1g3ERymw1LT02FXLHZKFaj39PdHWiiBBy6ELGXIMrUCsh0njU1OLfJyqa37eFt-pFdNj~Glth7EQQGRwWM5VgHz5A7PjMDtwpB1vThqzXgpcnJKchVzeP1mThuIyszWmBlqgtBnLbcmiV-B88pjBUNxp6nvJK~EPLNKadO8WW9zBVE96VDSpb57niRb6IGXhlx-jWCKvWMkN7Lsd9XzhSieY0sd1rWkFGwaA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Lfc−/− DCs exhibit reduced contractile responses. (a) Left: Cell outlines of Lfc+/+ (left) and Lfc−/− (right) DCs migrating within a collagen network along a soluble CCL19 gradient. Scale bar, 10 µm. Right: Graph shows the lengths of cells migrating within a collagen network of n = 85 individual 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 unpaired Student’s t test; ***, P ≤ 0.001. (b) Levels of active RhoA of Lfc+/+ and Lfc−/− cells were determined by luminometry showing the mean intensities ± SD from N = 3 experiments. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. (c) Levels of MLC phosphorylation (pMLC) in Lfc+/+ and Lfc−/− DCs assessed by Western blot analysis. Cells were treated with the indicated compounds (DMSO, CCL21, nocodazole [Noco.], or Y27632 together with nocodazole [Y/N]). (d) Mean fluorescence intensity of phospho-MLC was normalized to GAPDH signal and shown as fold increase relative to DMSO control ± SD. Blots are representative of N = 3 experiments. Annotation above columns indicates results of two-way ANOVA; *, P ≤ 0.1. r.u., relative units. (e) Centrosome localization relative to the nucleus in Lfc−/− DCs migrating under agarose assessed by α- and γ-tubulin costaining (n = 117 cells from N = 2 experiments ±SD). (f) Centrosome position relative to the nucleus of Lfc−/− DCs migrating within a path choice assay. Shown are mean frequencies of n = 49 cells from N = 2 experiments ±SD. cho., choice. (g) MT nucleation from centrosomal origin as determined by α- and γ-tubulin costaining. Blue line indicates the position of the nucleus. Scale bars, 10 µm. (h) Intensity line profiles across the highest γ-tubulin (tub.) signal along the left-right axis (dashed line in g). The purple line indicates γ-tubulin signal intensity. The black line indicates α-tubulin signal distribution. (i) Path choice preference of Lfc+/+ and Lfc−/− DCs migrating within a path choice assay. Shown are mean frequencies of Lfc−/− (n = 49 cells from N = 2 experiments) and Lfc+/+ (n = 79 cells from N = 3 experiments) DCs ±SD. (j) Frequency of cell rupturing events of Lfc+/+ (n = 73 cells from N = 3 experiments) and Lfc−/− (n = 128 cells from N = 3 experiments) DCs while migrating within single constriction–containing microchannels ±SD. (k) Migration speed of nocodazole-treated cells within straight microchannels of n = minimum of 80 cells per condition from N = 5 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. (l) Migratory persistence of nocodazole-treated Lfc+/+ and Lfc−/− DCs within straight microchannels of n = minimum of 80 cells per condition from N = 5 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 ≤ 0.01. n.s., not significant.
