Fig. 2: Loss of Swip-1 impairs lamellipodia formation and cell migration of macrophages.

a Schematic overview of the swip-1 gene locus. Exons encoding parts of distinct domains are indicated, EF domains in green and coiled-coil region in orange. The target sequence for CRISPR/Cas9 gene modification and generated swip-1 deletions are depicted. b Loss of swip-1 mutants were validated by Western blot analysis. Lysates from ten fly heads of wild-type and different mutant flies were analyzed. Non-specific bands (upper bands) serve as a loading control. All obtained mutants were validated once, hereinafter used swipΔ1 mutant flies were checked regularly in Western blot analysis of head lysates. c, d Confocal images of c wild-type and d swip-1 mutant macrophages were co-stained with phalloidin (white) and DAPI (blue). Scale bars 10 µm. e Quantification of lamellipodia width at the leading edge, n = WT: 64, swip-1 mutant: 61 cells of two independent experiments (P value: 0.012). f Circularity was measured using Image J shape descriptors, n = WT: 180, mutant: 192 (P value: >0.001), rescue: 209 cells (P value: 0.892). g Rescue of swip-1 mutant macrophages re-expressing a Swip-1-eGFP fusion were co-stained with phalloidin (white) and DAPI (blue). The arrow marks mutant cells lacking Swip-1-eGFP that still show an irregular cell shape. h, i Spinning disk microscopy live imaging of randomly migrating macrophages in prepupae with indicated genotypes were tracked by using Imaris 9.3. Track speed mean is color-coded (0.06 to 6 µm/min) (h) wild type and (i) overexpression of a Swip-1-eGFP (OE) in a wild-type background, j swip-1 mutant, (k) rescue with a Swip-1-eGFP, and l rescue with a Swip-1-D82A/D118A-eGFP, respectively. m Quantification of track speed mean, WT: n = 114 tracks, OE: n = 133 tracks (P value: 0.006), swip-1 mutant: n = 206 tracks (P value: <0.001), rescue WT: n = 166 tracks (P values: 0.002 to WT and <0.001 to mutant), rescue SwipD82A/D118A: n = 136 tracks (P values: 0.594 to WT and <0.001 to mutant). e, f, m Boxes indicate 50% (25–75%) and whiskers (5–95%) of all measurements, with black lines depicting the medians. For statistical analysis, two values indicated by connecting black lines were compared with two-sided Mann–Whitney test, P value: 0.12 (ns), 0.033 (*), 0.002 (**), <0.001 (***). n Frames of spinning disk microscopy video of migrating swip-1 mutant macrophages re-expressing Swip-1-D82A/D118A-eGFP. Images were taken every 20 s for 30 min. A white arrow indicates the direction of movement; yellow arrowheads mark the lamellipodium. Swip-1-D82A/D118A-eGFP localizes to the cell cortex but not to the protruding lamellipodium (see also Supplementary Video M3). Scale bar 10 µm. All images shown are representative of at least three independent experiments unless otherwise specified.