Fig. 5

Afp18^G blocks cell blebbing without affecting lamellipodia and filopodia formation.

(a) Bleb formation analysed at 30% epiboly (4.7 h.p.f.) by time series of embryos injected with 0.5 pg per embryo of Afp18^G NxN mRNA (upper two rows) or Afp18^G mRNA (lower two rows). Embryos were co-injected with mRNA encoding Lifeact-GFP (100 pg per embryo) labelling the F-actin cytoskeleton. DIC images are shown in the upper row and corresponding epifluorescence images in the lower row. Black arrowheads indicate spherical membrane protrusions of a blastomere, known as bleb formation, which are initially devoid of actin (white arrowheads). In contrast, blastomeres of Afp18^G-injected embryos show significantly less blebbing and blebs appear smaller. Graph shows the quantification of bleb formation of blastomeres in Afp18^G mRNA compared with Afp18^G NxN control injected embryos (0.5 pg per embryo) during a time series (frames: 10-s intervals for 300s; Afp18^G NxN n=75; Afp18^G n=75 cells analysed of three different embryos, 25 blastomeres each). Values are average±s.e.m.; statistical significance was analysed using Mann–Whitney test. Scale bar, 10 µm. (b) Protrusive activity analysed in time series of Afp18^G NxN (upper two rows) or Afp18^G (lower two rows) mRNA (0.5 pg per embryo each) injected embryos at 30% (4.7 h.p.f.). Embryos were co-injected with mRNA encoding Lifeact-GFP (100 pg per embryo) labelling the F-actin cytoskeleton. DIC images are shown in rows 1 and 3 and corresponding fluorescent image in the rows 2 and 4. Both Afp18^G NxN and Afp18^G mRNA-injected embryos show blastomeres forming sheet-like membrane protrusions resembling lamellipodia, filled with actin bundles and actin branches (arrows). Scale bar, 10 µm. Quantification shows the percentage of analysed blastomeres forming blebs without actin, blastomeres forming sheet-like membrane protrusions with actin or blastomeres without membrane protrusion activity during the captured time series (Afp18^G NxN n=41; Afp18^G n=37 cells analysed from three different embryos). The statistical significance (P=0.0091) of differences between the proportion of Afp18^G NxN and Afp18^G blastomeres showing these specific behaviours was computed using the Kruskal–Wallis test. The analysis revealed that the distribution of cell behaviour types indeed differs significantly, caused by changes in blebbing activity but not in lamellipodia formation. DIC, differential interference contrast.