Handling Editor: David Glover
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Figures
- Fig. 1.
synd shows defects in cap and furrow remodeling and F-actin dynamics. (A) Imaging Drosophila syncytial NCs using an inverted microscope (left). NC schematic (right) is rotated by 180° with apical at the top to represent the orientation in sagittal views. Interphase embryos have apical centrioles and short furrows, and metaphase embryos have lateral centrosomes and long furrows. Dashed line indicates the plane of membrane shown in the top panel. (B–D) synd embryos expressing tGPH show significantly increased NC12 cap area (B,C) and significantly shorter furrow length (B,D). (E,F) Peripheral LifeAct–GFP intensity in synd embryos is significantly higher than in controls. Graphs show mean±s.e.m. for n=3 embryos, with three caps quantified per embryo. ***P=0.001 (one-way ANOVA and Tukey's multiple comparison test). Scale bars: 10 μm.
- Fig. 2.
Apical protrusion remodeling is defective in synd. (A) LifeAct–GFP-labeled protrusions in caps in control and synd NC12 embryos by TIRFM. Scale bar: 5 μm. (B) Relative frequency distributions of protrusion lengths show significantly longer interphase protrusions (0.86±0.3 μm, mean±s.d.) compared to metaphase protrusions (0.67±0.19 μm) in controls (***P<0.001) but not in synd (0.81±0.21 μm, interphase; 0.75±0.21 μm, metaphase; ns, P>0.05). n=100 protrusions per cap, three caps per embryo for three embryos. Statistical significance was tested using a one-way ANOVA and Tukey's multiple comparis on test. (C,D) STED microscopy of phalloidin-stained caps and furrows in control and synd NC12 embryos. Representative images are shown in C. Longer protrusions are observed during interphase and not during metaphase for control embryos. Long protrusions are present during metaphase and furrows are missing in 100% of synd embryos (n=10 embryos each). Protrusion lengths (mean±s.d.) are significantly different between interphase and metaphase for control embryos but not for synd embryos (D). n=3 embryos, ∼50 protrusions/embryo. ***P<0.001; ns, P≥0.05 (two-tailed Mann–Whitney test). Scale bar: 3 μm. (E) Cap area increases and protrusion (red arrow) lengths decrease from interphase to metaphase in controls. In synd, the cap area is larger during interphase, apical protrusions remain and furrow length (blue arrow) is shorter than in controls during metaphase.
- Fig. 3.
arpC1/+;synd cap and furrow dynamics are similar to controls. (A–C) Cap area (A,B) and furrow length (A,C) of arpC1/+;synd is comparable to that of controls (data for controls is repeated from Fig. 1B,C). Data are mean±s.e.m. of n=3 embryos, with three caps measured per embryo. ***P=0.001 (one-way ANOVA and Tukey's multiple comparison test). Scale bar:10 μm. (D) Apical protrusions imaged by TIRFM in NC12 arpC1/+;synd embryos. Scale bar: 5 μm. (E) Relative frequency distribution of protrusion lengths shows a significant difference between interphase (0.72±0.36 μm, mean±s.d.) and metaphase (0.6±0.3 μm) in arpC1/+;synd (***P<0.001; one-way ANOVA and Tukey's multiple comparison test) similar to controls (Fig. 2B). n=100 protrusions per cap, for three caps per embryo in three embryos. (F) STED microscopy of phalloidin in caps and furrows of arpC1/+;synd in NC12. Long protrusions are absent and furrows are present in arpC1/+;synd (89%, n=9 embryos). Scale bar: 3 μm. (G) Protrusion lengths (mean±s.d.) are significantly different between interphase and metaphase in arpC1/+;synd, as seen for controls, and unlike synd (Fig. 2D). n=3 embryos with ∼50 protrusions measured per embryo, ***P<0.001 (two-tailed Mann–Whitney test). (H) arpC1/+;synd shows decreased protrusion length and increased furrow lengths in metaphase.
- Fig. 4.
Myosin II is reduced in synd. (A) Dlg is unaffected and Zipper is reduced and remains at tricellular junctions in synd (100%, n=15 embryos; red arrowhead) compared to control (100%, n=20 embryos) in metaphase, Zipper levels are rescued in arpC1/+;synd (87.5%, n=16 embryos). Scale bar: 10 μm. (B) Sqh–GFP in control, synd and arpC1/+;synd. Representative image (9 min) with increased contrast shows Sqh–GFP dissociation from the membrane in control and arpC1/+;synd, and not in synd (red arrowhead). Scale bar: 10 μm. (C) Relative Myosin II intensity (assayed using Sqh–GFP) versus time. Data are mean±s.e.m. of n=3 embryos with three caps quantified per embryo. ***P=0.001 (one-way ANOVA and Tukey's multiple comparison test). (D) Myosin II intensity (assayed using Sqh–GFP) in the indicated strains. n=3 embryos with three caps measured per embryo. ***P<0.001; ns, not significant (non-parametric Mann–Whitney U-test).
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