Supplemental Figures
Supplementary Movie 1
SCAR mutant hemocytes migrate more slowly from both the anterior and posterior of the embryo compared to wild types. 3 hour long timelapse movies of wild type (w;srp-Gal4,UAS-red stinger) and SCARΔ37 mutant (w;
SCARΔ37,srp-Gal4;crq-Gal4,uas-red stinger) embryos with red stinger-labelled hemocytes from comparable points in stage 12. Each frame is a maximum projection, with 2 minutes between each timepoint. Scale bar represents 50
m. Movies correspond to maximal projections through time shown in Figures
1g and h.
Supplementary Movie 2
Filopodia-like structures are motile in SCAR mutant hemocytes. 1 hour movie showing maximum projection of mCherry-moesin-labelled hemocytes in a w;SCARΔ37,srp-Gal4;pxn-Gal4,UAS-mCherry-moesin embryo at stage 16 of development. Actin-rich ‘spindly’ protrusions are highly motile. Contrast has been enhanced in each frame owing to photobleaching of mCherry-moesin in
ImageJ; images were collected at a rate of 1 per minute on a Leica DMI
6000B UltraVIEW VoX spinning disk confocal imaging system with an HCX
PLANAPO 40X/1.30 oil immersion objective using Volocity software (Perkin
Elmer).
Supplementary Figure 1 Loss of WASp has no effect on lamellipodia in a SCAR mutant background.
(a-b) Hemocyte morphology was very similar between hemocytes in SCAR mutant (w;SCARΔ37,srp-Gal4,UAS-GFP) (a) and SCAR;WASp double mutant embryos (w;SCARΔ37,srp-Gal4,UAS-GFP;WASpEY06238) (b). (c) Quantification of lamellipodial areas revealed no significant differences between the two genotypes (ns=not significant; t-test, n≥30 hemocytes taken from ≥9 embryos per genotype), suggesting that WASp neither acts redundantly with SCAR in the formation of residual lamellipodia, nor sequesters the Arp2/3 complex from any remaining maternal SCAR. Scale bars represent 20 m.
Supplementary Figure 2
Loss of WASH function does not affect hemocyte dispersal and exhibits no effect on lamellipodia in a SCAR mutant background. (a-b) Red stinger- labelled hemocytes in stage 13/14 wild type (w;;crq-Gal4,UAS-red stinger) (a) and WASH mutant embryos (w;WASHΔ185;crq-Gal4,UAS-red stinger) (b). (c)
Bar graph of mean numbers of segments lacking hemocytes at stage 13/14
(ns=not significant; Kruskal-Wallis test, Dunn’s post-test, n>30 embryos per genotype); error bars represent standard deviation. (d-e) Hemocytes in both wild type (w;;crq-Gal4,UAS-GFP) (d) and WASH mutant embryos
(w;WASHΔ185;crq-Gal4,UAS-GFP) (e) exhibited large lamellipodia (there was no significant difference in lamellipodial area, data not shown). (f) Hemocytes in SCAR,WASH double mutants (w;SCARΔ37,srp-Gal4,WASHΔ185;crq-
Gal4,UAS-GFP) were indistinguishable from those in SCAR mutants (there was no difference in lamellipodial areas, data not shown). (g) Movies of red stinger-labelled hemocytes revealed there was no difference between SCAR and SCAR,WASH double mutant embryos, whilst there was only a small reduction in speed between WASH mutants and the appropriate wild type controls. Genotypes in (g) were (left to right) w;srp-Gal4;crq-Gal4,UAS-red stinger, w;SCARΔ37,srp-Gal4;crq-Gal4,red stinger, w;SCARΔ37,srp-
Gal4,WASHΔ185;crq-Gal4,UAS-red stinger, w;WASHΔ185;crq-Gal4,UAS-red stinger and w;;crq-Gal4,UAS-red stinger. Scale bars represent 50 m (a-b),
10 m (d-e) and 20 m (f).
Supplementary Figure 3
SCAR mutant embryos do not exhibit a pronounced increase in apoptosis and their hemocytes do not appear to be vacuolated due to cell autonomous death. (a-b) Acridine orange staining of wild type (a) (w;srp-Gal4;crq-
Gal4,uas-GFP) and SCARΔ37 mutant (b) (w;SCARΔ37,srp-Gal4;crq-Gal4,uas-
GFP) embryos showed no gross increase in apoptosis at indicated stages of development; anterior is to the right, with orientation as noted. (c-e) Acridine orange (ao, green) staining of red stinger-labelled hemocytes (red nuclei) in wild type (c) (w;srp-Gal4,uas-red stinger), SCARΔ37 (d) (w;SCARΔ37,srp-
Gal4,UAS-red stinger;H99/TM6b-P{Dfd-GMR-nvYFP}) and SCARΔ37;H99 double mutant embryos (e) (w;SCARΔ37,srp-Gal4,UAS-red stinger;H99) revealed that acridine orange has not entered the nucleus as might be expected if these cells were undergoing autophagic cell death. (f-g)
Expression of p35 specifically in hemocytes failed to prevent their vacuolation or restore lamellipodia as visualised via GFP co-expression at stage 15, suggesting that vacuoles do not represent dying hemocytes, nor are the hemocytes themselves dying by apoptosis; (f) is a zoomed region from another embryo revealing spindle-like processes remain in presence of p35 expression. Scale bars represent 50 m (a-b), 20 m (c-f) and 10 m (g).
Supplementary Table 1
Genotypes of fly embryos used in Figures 1-7