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Fig. 7. Impaired lamellipodia formation and defective actin reorganisation in TM9SF4 mutant macrophages. Circulating plasmatocytes were isolated from wild-type (A,C) or mutant TM9SF4 (B,D) third instar larvae and allowed to spread for 15 minutes in a glass coverslip chamber. (A,B) Phase contrast. (C,D) Reflection interference contrast microscopy. Arrowhead indicates the loss of adhesive belt; arrow indicates the white area that represents more distant contacts. (E-I) Confocal analysis of actin network in isolated larval macrophages. Texas-Red-phalloidin fluorescent labelling revealed polymerised actin (E,H) and nuclei were stained with Hoechst 33258 (F,I); overlays are shown in G,J. Control cells are regularly sized and round (E-G), whereas most TM9SF4 mutant cells have a larger area and differentiate long actin-stained filopodia (H-J). (K) The surface of the cytoskeleton network was calculated from 500-1000 cells. Mutant TM9SF4 cells were 2.3-fold larger than Rev45 control cells (P<0.0001, Student's t-test). Cell size was partially rescued by expression of TM9SF4 cDNA in the haemocyte lineage. A significant difference between TM9SF4 mutant and srpGal4; TM9SF4;UAS-TM9SF4/+ rescued plasmatocytes was found (P<0.004, Student's t-test) (larvae raised at 18°C). Scale bars: 10 µm.
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