Fig. 3

Fig. 3

Rac2 Activity Is Required for Neutrophil Motility and Polarity In Vivo

(A) Tracking of neutrophil random migration in the head in Rac2 and D57N larvae (lateral view). Scale bar represents 50 μm.

(B) Quantification of 3D speed of neutrophil random motility in mesenchymal tissues of the head of Rac2 or D57N larvae. n = 38 (Rac2) and 45 (D57N). p < 0.001, two-tailed Mann-Whitney U test.

(C) Quantification of 3D speed of neutrophil random motility in mesenchymal tissues in the head of Ctrl and Rac2 morphants. n = 25 (Ctrl MO) and 22 (Rac MO). p < 0.001, two-tailed Mann-Whitney U test.

(D and E) Tg(mpx:GFP-PH-Akt) was crossed to Rac2 (D) and D57N (E) lines to visualize the localization of PI(3,4,5)P₃-PI(3,4)₂ in neutrophils randomly migrating in mesenchymal tissues of the head. Ratiometric images were generated using mCherry as a volumetric control. Still images from representative movies of randomly migrating neutrophils in the head are shown. Arrows, pseudopods in (D) and protrusions in (E).

(F) Tg(mpx:GFP-UtrCH) was crossed to Rac2 and D57N lines to visualize the localization of stable F-actin. Still images from representative movies of randomly-migrating neutrophils in the head are shown. Upper, arrow, uropod; arrowhead, retracting pseudopods. Lower, arrow, protrusion. Scale bar represents 5 μm (D-F).

(G) Schematics of altered cell polarity in D57N neutrophils. PI(3,4,5)P₃-PI(3,4)₂ primarily localizes to the pseudopods in Rac2 neutrophils. D57N neutrophils form transient protrusions that are enriched for PI(3,4,5)P₃-PI(3,4)₂, but fail to form functional pseudopods. Stable F-actin is polarized and enriched in the uropod in Rac2 neutrophils but is present in protrusions and is not polarized in D57N neutrophils.

See also Figure S2, Movie S2, Movie S3, Movie S4, and Movie S5.