SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila

Abstract

In vitro studies have shown that SCAR/WAVE activates the Arp2/3 complex to generate actin filaments, which in many cell types are organised into lamellipodia that are thought to have an important role in cell migration. Here we demonstrate that SCAR is utilised by Drosophila macrophages to drive their developmental and inflammatory migrations and that it is regulated via the Hem/Kette/Nap1-containing SCAR/WAVE complex. SCAR is also important in protecting against bacterial pathogens and in wound repair as SCAR mutant embryos succumb more readily to both sterile and infected wounds. However, in addition to driving the formation of lamellipodia in macrophages, SCAR is required cell autonomously for the correct processing of phagocytosed apoptotic corpses by these professional phagocytes. Removal of this phagocytic burden by preventing apoptosis rescues macrophage lamellipodia formation and partially restores motility. Our results show that efficient processing of phagosomes is critical for effective macrophage migration in vivo. These findings have important implications for the resolution of macrophages from chronic wounds and the behaviour of those associated with tumours, because phagocytosis of debris may serve to prolong the presence of these cells at these sites of pathology.

Figure 1

SCAR is required by migrating macrophages for their developmental dispersal and interacts genetically with Pvr. (a–d) Lateral views of stage 13/14 embryos immunostained to show distribution of GFP-labelled hemocytes. Hemocytes migrate from the head to form a single line atop the VNC in wild-type (WT) embryos (a); shown between arrows. Hemocytes failed to migrate as far in SCAR^Δ37 zygotic mutants (b) or when dominant-negative SCAR (dn SCAR) was specifically expressed in hemocytes (c). Regions between arrowheads indicate absence of hemocytes (b and c). Expression of wild-type SCAR specifically in hemocytes in a SCAR^Δ37 background (SCAR rescue) rescued this defect (d). (e) Quantification showing mean number of empty segments in fixed embryos at st13/14; each SCAR mutant, with the exception of SCAR rescue embryos, was significantly different from wild types (P≤0.001, Kruskal–Wallis test, Dunn's multiple comparison post test, n≥29 embryos per genotype), whereas hemocyte-specific expression of the anti-apoptotic gene p35 in a SCAR^Δ37 mutant background (SCAR+p35) failed to rescue. N.B. 2x dn SCAR represents a 50:50 mix of embryos with hemocytes expressing from 1 or 2 copies of UAS-dn SCAR (see Materials and methods section). (f) Quantification of mean position of lead hemocytes for anterior and posterior populations from fixed embryos; where hemocytes were present all along the VNC they were assumed to have met at segment 7.6, a value that corresponds to the mean meeting point taken from ten movies of WT embryos. (g and h) Projections of all time points of 3 h-long movies of WT and SCAR^Δ37 mutant embryos with red stinger-labelled hemocytes (see also Supplementary Movie 1) revealed that hemocytes from both posterior and anterior populations were retarded in SCAR^Δ37 mutants; hemocytes of these two populations met at the arrowhead in (g); representative of at least three movies per genotype. (i) Mean number of empty segments per embryo in SCAR^Δ37/+ and Pvr¹/+ heterozygotes and SCAR^Δ37/ Pvr¹ transheterozygotes showing genetic interaction between Pvr¹ and SCAR^Δ37. Each genotype is significantly different from the others (P≤0.05, Kruskal–Wallis test, Dunn's multiple comparison post test, n≥35 embryos per genotype). Anterior is to the right and ventral is up in all images; scale bars represent 50 μm; error bars represent the S.D.

Figure 2

SCAR drives inflammatory migration of hemocytes. (a and b) Stage 15 wild-type (a) and SCAR^Δ37 mutant embryos (b) with GFP-labelled hemocytes (green) were wounded and imaged 1 h later (purple borders show wound position). (c and d) Scatter (c) and box and whisker plots (d) reveal that significantly fewer hemocytes were present per μm² of wound area in SCAR^Δ37 mutants (P≤0.001, t-test, n≥20 embryos per genotype). (e,e') Box and whisker plots of hemocyte speed (e) and directionality (e') show that SCAR^Δ37 mutant hemocytes migrated to wounds significantly more slowly (P≤0.001, t-test, 50 WT and 20 SCAR^Δ37 tracks taken from seven movies of each genotype), but as directionally as wild types (NS, not significant). Scale bars represent 20 μm. Lines represent the median, whereas boxes and whiskers show the interquartile and 2.5–97.5 percentile ranges, respectively, in these and all subsequent box and whisker plots

Figure 3

SCAR drives hemocyte migration and is necessary for normal hemocyte morphology. (a–c) Hemocyte tracks representing migration for 30 min at stage 15 in WT (a), SCAR^Δ37 (b) and SCAR rescue embryos (c); arrows show position of ventral midline. (a'–c') Morphology of GFP-labelled hemocytes at stage 15 on the ventral midline; arrowheads illustrate large lamellipodia, asterisks indicate spindly protrusions. (a”–c”) F-actin morphology of hemocytes at the ventral midline shown via mCherry-moesin expression. (d and e) Hemocytes expressing GFP and dn SCAR from one UAS construct showed a mild reduction in lamellipodia (d), but a more severe phenotype was frequently seen in embryos that expressed dn SCAR from either 1 or 2 copies of the UAS insertion (e). (f–h) Box and whiskers plots showing quantification of hemocyte speeds (f; n≥100 hemocytes from ≥7 embryos per genotype) and lamellipodial area (g; n≥25 hemocytes from ≥15 embryos per genotype) and numbers of vacuoles per hemocyte (h; n≥34 hemocytes from ≥13 embryos per genotype); each category is significantly different from the rest (P≤0.05, one-way ANOVA, Bonferroni's post test), except where indicated (NS not significant). All scale bars represent 20 μm

Figure 4

SCAR mutant hemocytes contain numerous apoptotic corpses and have defective phagosome processing. (a and b) Maximum projections (a) and zooms of single confocal slices from indicated box region (a') reveal that vacuoles within mCherry-moesin-labelled (ch moe) wild-type (WT) hemocytes appear as regions excluding this F-actin marker (asterisks indicate examples); co-staining with acridine orange (ao=red and/or green spheres, arrowheads indicate examples; red=ch moe) shows that the majority of these vacuoles contain apoptotic corpses (b). (c and d) H99 mutants neither contained vacuoles (c–c'), nor stained with acridine orange (d). (e and f) SCAR^Δ37 mutants were highly vacuolated (e–e') and the majority of vacuoles stained positively for acridine orange (f). (g) Stage 15 hemocytes co-expressing dominant-negative Rab5 and GFP also became highly vacuolated, although they retained lamellipodia (indicated by arrows in zoomed region). (h) Quantification revealed that the mean fraction of vacuoles positively labelled with LysoTracker red within hemocytes per embryo was significantly reduced in both SCAR^Δ37 homozygotes and embryos that contained dn Rab-expressing hemocytes at stage 15 compared with wild types (P≤0.01, one-way ANOVA, Bonferroni's post test, n≥12 embryos per genotype); error bars represent the S.D.; scale bars show 10 μm. NS, not significant

Figure 5

The SCAR complex component Hem is also required for lamellipodial formation and hemocyte migrations. (a) Immunostained stage 13/14 Hem^J4-48 mutant embryo with GFP-labelled hemocytes; hemocytes failed to occupy the entire length of the VNC (region bounded by arrowheads). (b) Quantification showing average number of segments devoid of hemocytes at stage 13/14 in Hem⁰³³³⁵ heterozygotes and Hem⁰³³³⁵ and Hem^J4-48 mutant embryos; error bars represent S.D.; each population is significantly different from the others (P≤0.01 Kruskal–Wallis test, Dunn's post test, n≥30 embryos per genotype). (c and d) Tracks of red stinger-labelled hemocytes migrating for 30 min over the VNC at stage 15 in wild-type (c) and Hem^J4-48 mutant embryos (d). (e) Box and whiskers plot showing average speed per hemocyte; speeds were significantly different (P≤0.0001, t-test, n≥92 hemocytes from six embryos per genotype). (f–k) GFP and GMA-labelled hemocytes at stage 15 at the ventral midline in indicated genotypes revealing that Hem is required for lamellipodia and its absence leads to vacuolation; anterior is up. Scale bars represent 50 μm (a), 20 μm (c and d, f-h) and 10 μm (i–k)

Figure 6

SCAR mutants exhibit defects in clearance of bacteria and damage repair and have enhanced susceptibility to pathogens. (a and b) Stage 15 embryos with GFP-labelled hemocytes (green) were injected with RFP-labelled E. coli (red) and imaged 1 h later (a and b), revealing a defect in phagocytosis in SCAR^Δ37 mutants (b) compared with wild types (a). (c) Bar graph showing mean ratio of phagocytosed E. coli:total E. coli in field of view at 1 h post-injection; there was a significant reduction in the amount of E. coli phagocytosed in SCAR^Δ37 mutants (P<0.01, Mann–Whitney test, n≥4 per genotype). (d) Bar graph showing mean viability of non-injected controls (NI) and embryos injected with PBS or ecc15; each column is significantly different (P<0.01, one-way ANOVA, Bonferroni's post test, n=3-4 with ≥100 embryos injected per experiment), except where indicated (NS), suggesting that SCAR^Δ37 mutants exhibit defects in repair of damage caused by injection and are more susceptible to ecc15-induced death. (d') Normalising according to the percentage of embryos that hatch following injection with PBS and then expressing this value relative to the WT+ecc15 condition shows that the enhanced lethality of SCAR mutants on injection with ecc15 was not a consequence of their increased susceptibility to damage (P<0.05, Student's t-test). Scale and error bars represent 5 μm and the S.D., respectively. NS, not significant

Figure 7

Removing apoptosis rescues lamellipodia and partially rescues speed in SCAR mutant hemocytes. (a–d) GFP expression in hemocytes at stage 15 in wild-type (a), SCAR^Δ37 (b), SCAR^Δ37;H99 double mutant (c) and H99 mutant (d) embryos revealed that the H99 deficiency rescued lamellipodia (arrows). Regions with reduced levels of GFP in cell bodies in (c) and (d) are nuclei (asterisks); scale bars represent 20 μm. (e) Bar graph showing mean number of segments lacking hemocytes at stage 13; error bars represent the S.D. SCAR^Δ37 mutants exhibited a significant defect compared with the other genotypes (P≤0.01; Kruskal–Wallis test, Dunn's multiple comparison post test, n≥15 embryos per genotype), whereas there were no significant differences amongst the remaining comparisons. (f–h) Box and whisker plots of hemocyte lamellipodial area (f; n>50 hemocytes from ≥12 embryos per genotype), speed (g; n≥90 hemocytes from ≥5 embryos per genotype) and rate of lamellipodial change per hemocyte per min (h; >12 hemocytes were analysed from three different movies) at stage 15. Asterisks indicate a significant difference between SCAR^Δ37 mutants and the other genotypes in (f) (P≤0.001) and with WT and SCAR;H99 in (h) (P≤0.01). P-values determined by one-way ANOVA and Bonferroni's post test (f–h)