Figure S1.
DC migration within diverse matrices to study the role of the MT cytoskeleton during cell migration. (a) Schematic representation of migration assays used in this study. Assays range from highly complex (top) and relatively uncontrollable geometries to very simple and precisely controllable PDMS-based structures (bottom). Complexity of the geometrical confinement correlates with dynamic shape changes of cells. Numbers of upward-facing arrows scale with high geometrical complexity and cell shape changes. Numbers of downward-facing arrows scale with low complexity. (b) Cell shape changes of a DC migrating in a collagen matrix along a soluble CCL19 gradient. Scale bar, 10 μm. (c) Dynamic cell shape changes are recapitulated during migration within a defined array of PDMS-based pillar structures. (d) MT nucleation from centrosomal origin determined by α- and γ-tubulin (tub.) staining. Right panel: The line profile of mean fluorescence intensities (MFI) along the purple line in the merged image is shown. Scale bar, 10 µm. (e) Determination of MTOC position by α- and γ-tubulin staining with respect to the nucleus. Black arrowheads indicate MTOC position. Mean ± SD of n = 256 cells from N = 3 experiments. (f) Double-reporter DC migrating under agarose along a soluble CCL19 gradient. Left panel: Cells migrating under agarose display a protrusive lamellipodium (lower panel: montage of boxed area) followed by a contractile trailing edge. Scale bar, 10 µm. Middle panel: EB3-mCherry (EB3-mChe.) localizes to the plus tips of tubulin-GFP–decorated MT filaments. Scale bar, 10 µm. Right panel: EB3-mCherry faithfully tracks growing MT filaments during DC migration. White arrowheads highlight the localization of EB3 signal at the tip of polymerizing tubulin filaments as the cell advances. Scale bar, 5 µm. (g) Automatically detected EB3 comets (cyan) overlaid on maximum intensity time projection (120 s) of an EB3-mcherry–expressing cell migrating under agarose. Lower panel: Quantification of MT growth events of front (gray) versus back (purple) directed MT tracks over a time period of 120 s of n = 7 cells. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Scale bar, 10 µm. (h) Time-course analysis of MT filament dynamics of migrating DCs expressing EMTB-mCherry. Upper panel: Indicates the leading edge area. The white arrow represents membrane protrusion, and the white arrowheads represent elongating MT filaments. Lower panel: Indicates the trailing edge area in which the purple arrow represents membrane retraction and purple arrowheads MT filament depolymerization. Red dashed lines indicate cell edges. Scale bar, 10 µm. (i) Acetylated-tubulin (ace. tub.) staining in DCs fixed while migrating under agarose. Only cells with the MTOC localized in front of the nucleus were analyzed. Levels of acetylation were assessed by measuring the mean fluorescence intensity of acetylated-tubulin along individual α-tubulin (a.-tub) filaments (n = 87 filaments per condition from N = 3 experiments) directed toward the front (gray) or back (purple). 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 ≤ 0.0001. Scale bar, 10 µm. (j) EB3-mCherry localization of control or PST-1–treated cells migrating under agarose along a soluble CCL19 gradient. The red box indicates the photoactivated area magnified on the right. Magnified regions show time projection of EB3-mCherry intensities after local photoactivation. Purple arrows indicate the direction of cell migration. Lower panel on the right indicates fluorescence intensity evolution upon photoactivation of control or PST-1–treated cells. The red line highlights the time point of the initial photoactivation. Scale bar, 10 µm. activ., activation; NT, non-treated; r.u., relative units.

DC migration within diverse matrices to study the role of the MT cytoskeleton during cell migration. (a) Schematic representation of migration assays used in this study. Assays range from highly complex (top) and relatively uncontrollable geometries to very simple and precisely controllable PDMS-based structures (bottom). Complexity of the geometrical confinement correlates with dynamic shape changes of cells. Numbers of upward-facing arrows scale with high geometrical complexity and cell shape changes. Numbers of downward-facing arrows scale with low complexity. (b) Cell shape changes of a DC migrating in a collagen matrix along a soluble CCL19 gradient. Scale bar, 10 μm. (c) Dynamic cell shape changes are recapitulated during migration within a defined array of PDMS-based pillar structures. (d) MT nucleation from centrosomal origin determined by α- and γ-tubulin (tub.) staining. Right panel: The line profile of mean fluorescence intensities (MFI) along the purple line in the merged image is shown. Scale bar, 10 µm. (e) Determination of MTOC position by α- and γ-tubulin staining with respect to the nucleus. Black arrowheads indicate MTOC position. Mean ± SD of n = 256 cells from N = 3 experiments. (f) Double-reporter DC migrating under agarose along a soluble CCL19 gradient. Left panel: Cells migrating under agarose display a protrusive lamellipodium (lower panel: montage of boxed area) followed by a contractile trailing edge. Scale bar, 10 µm. Middle panel: EB3-mCherry (EB3-mChe.) localizes to the plus tips of tubulin-GFP–decorated MT filaments. Scale bar, 10 µm. Right panel: EB3-mCherry faithfully tracks growing MT filaments during DC migration. White arrowheads highlight the localization of EB3 signal at the tip of polymerizing tubulin filaments as the cell advances. Scale bar, 5 µm. (g) Automatically detected EB3 comets (cyan) overlaid on maximum intensity time projection (120 s) of an EB3-mcherry–expressing cell migrating under agarose. Lower panel: Quantification of MT growth events of front (gray) versus back (purple) directed MT tracks over a time period of 120 s of n = 7 cells. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Scale bar, 10 µm. (h) Time-course analysis of MT filament dynamics of migrating DCs expressing EMTB-mCherry. Upper panel: Indicates the leading edge area. The white arrow represents membrane protrusion, and the white arrowheads represent elongating MT filaments. Lower panel: Indicates the trailing edge area in which the purple arrow represents membrane retraction and purple arrowheads MT filament depolymerization. Red dashed lines indicate cell edges. Scale bar, 10 µm. (i) Acetylated-tubulin (ace. tub.) staining in DCs fixed while migrating under agarose. Only cells with the MTOC localized in front of the nucleus were analyzed. Levels of acetylation were assessed by measuring the mean fluorescence intensity of acetylated-tubulin along individual α-tubulin (a.-tub) filaments (n = 87 filaments per condition from N = 3 experiments) directed toward the front (gray) or back (purple). 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 ≤ 0.0001. Scale bar, 10 µm. (j) EB3-mCherry localization of control or PST-1–treated cells migrating under agarose along a soluble CCL19 gradient. The red box indicates the photoactivated area magnified on the right. Magnified regions show time projection of EB3-mCherry intensities after local photoactivation. Purple arrows indicate the direction of cell migration. Lower panel on the right indicates fluorescence intensity evolution upon photoactivation of control or PST-1–treated cells. The red line highlights the time point of the initial photoactivation. Scale bar, 10 µm. activ., activation; NT, non-treated; r.u., relative units.

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