Figure 4.
The RhoA GEF Lfc associates with MTs and accumulates at sites of retraction. (a) Polarized distribution of Lfc-GFP during DC migration. Maximum intensity time projection (proj.) of a double-fluorescent reporter cell expressing Lfc-GFP and EB3-mCherry over 8.5 min. Diffuse Lfc-GFP accumulation is highlighted in the trailing edge (purple arrowheads) and in retracting protrusions (orange arrowheads). Scale bar, 10 µm. (b) Enrichment of non-filamentous Lfc-GFP or EB3-mCherry signal in the rear versus the front of migrating cells. Maximum intensity time projection over 100 s. Scale bar, 5 µm. Lower panel: Relative enrichment of non-filamentous fluorescence signal intensities of Lfc-GFP and EB3-mCherry in the rear versus the front of n = 16 cells from N = 3 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 ≤ 0.0001. (c) Differential localization of Lfc-GFP and MLC-RFP in protrusive (front, gray box) or contractile (back, purple box) area. Scale bar, 10 µm. (d) Co-localization (co-loc.) between Lfc-GFP and MLC-RFP; hot colors indicate strong co-localization, and cold colors specify exclusion. Right graph shows the correlation of co-localization over time. Boxed regions in c indicate exemplary regions used for the analysis of n = 8 cells ± SD. Co-localization was determined separately in actively protruding (gray box) and retracting (purple box) areas. (e) Distribution of Lfc-GFP and EB3-mCherry during migration within a pillar array. Time course of protrusion formation and protrusion retraction of a migrating fluorescent reporter cell. Dashed red line indicates cell outline; solid red line, individual pillars. Orange arrowhead indicates Lfc-GFP accumulation during protrusion retraction. Scale bar, 5 µm. See also Video 7.

The RhoA GEF Lfc associates with MTs and accumulates at sites of retraction. (a) Polarized distribution of Lfc-GFP during DC migration. Maximum intensity time projection (proj.) of a double-fluorescent reporter cell expressing Lfc-GFP and EB3-mCherry over 8.5 min. Diffuse Lfc-GFP accumulation is highlighted in the trailing edge (purple arrowheads) and in retracting protrusions (orange arrowheads). Scale bar, 10 µm. (b) Enrichment of non-filamentous Lfc-GFP or EB3-mCherry signal in the rear versus the front of migrating cells. Maximum intensity time projection over 100 s. Scale bar, 5 µm. Lower panel: Relative enrichment of non-filamentous fluorescence signal intensities of Lfc-GFP and EB3-mCherry in the rear versus the front of n = 16 cells from N = 3 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 ≤ 0.0001. (c) Differential localization of Lfc-GFP and MLC-RFP in protrusive (front, gray box) or contractile (back, purple box) area. Scale bar, 10 µm. (d) Co-localization (co-loc.) between Lfc-GFP and MLC-RFP; hot colors indicate strong co-localization, and cold colors specify exclusion. Right graph shows the correlation of co-localization over time. Boxed regions in c indicate exemplary regions used for the analysis of n = 8 cells ± SD. Co-localization was determined separately in actively protruding (gray box) and retracting (purple box) areas. (e) Distribution of Lfc-GFP and EB3-mCherry during migration within a pillar array. Time course of protrusion formation and protrusion retraction of a migrating fluorescent reporter cell. Dashed red line indicates cell outline; solid red line, individual pillars. Orange arrowhead indicates Lfc-GFP accumulation during protrusion retraction. Scale bar, 5 µm. See also Video 7.

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