Kin-5[G394tev]-GFP is cleaved by TEV protease. (A) Image of living embryos expressing Kin-5[G394tev]-GFP with or without TEV protease expressed in a striped pattern. Scale bar: 50 µm. Region marked by squares in yellow are shown in high magnification on the right (scale bar: 10 µm). Quantification of GFP signal along the anterior–posterior body axis (line in green) is shown in graph below. Region of TEV expression is highlighted in red. (B–D) TEV protease or buffer was injected into syncytial embryos mutant for Kinesin-5 and expressing Kin-5[G394tev]-GFP. (B) Images from time-lapse recordings in minute:second. (C) GFP fluorescence following TEV injection. Plotted are the mean (solid) and s.d. (dashed). N, number of quantified embryos for each experimental condition. (D) Images of living embryos before and 30 min after injection. Scale bars: 10 µm. (E) Western blot with extracts from embryos 30 min after injection with TEV or buffer probed with Kinesin-5 and α-tubulin antibodies.

Phenotype of Kin-5[G394tev]-GFP cleavage by TEV protease. (A) Images from time-lapse recordings of embryos mutant for Kinesin-5 expressing the Kin-5[G394tev]-GFP, His2Av-GFP, α-Tubulin–mCherry transgene and injected with TEV protease. Arrowhead indicates embryo undergoing defective mitosis. (B) Images from the control experiment. TEV protease was injected into the embryo expressing His2Av-GFP, α-Tubulin–mCherry, without the Kin-5 guillotine. Schematic interpretation of mitotic stages are shown below each panel; red, nuclei/chromosomes; green, microtubules. Scale bars: 10 µm. (C) Proportion of abnormal spindles after TEV injection. Data are mean±s.d. N=148 spindles in 3 embryos for ΔKin-5, Kin-5-tev-GFP, 348 spindles in 3 embryos for Kin-5-tev-GFP and 1384 spindles in 3 embryos for wild type. Source data are listed in Table S1.

Kinesin-5 is important for nuclear positioning in interphase. (A) Projected image of an embryo expressing Histone 2Av from selective plane illumination microscopy in side view and cross section (position indicated by lines in blue). Magnified sections illustrate the interactions between the nuclei and between nuclei and cortex, respectively. Dots in yellow indicate centrosome pairs. (B) Image of living embryo expressing Kin-5–GFP (apical position) Scale bar: 5 µm. (C) Images from time-lapse recording of embryos mutant for Kinesin-5 expressing the Kin-5[G394tev]-GFP transgene and co-injected with TEV protease and fluorescent labeled Histone H1. The green and red arrowheads indicate two nuclei that underwent a nuclear division cycle. After successful daughter nuclei separation, the two non-daughter nuclei fuse as indicated by yellow arrowhead. Scale bar: 10 µm. (D) Nuclei labeled with injected Histone H1. Embryos (Kinesin-5 mutant with Kin-5[G394tev]-GFP transgene) injected with buffer or TEV protease and fluorescently labeled Histone H1 to label nuclei. Scale bar: 10 µm. (E) Segmented images from D. Color coding for the number of neighboring cells is indicated. The nuclei labeled with empty circles were not included in the calculation. (F) Proportion of nuclei according to number of neighbors and (G) irregularity of nuclear arrangement in embryos injected with buffer or TEV protease. The irregularity parameter σ/μ in Kinesin-5-depleted and control embryo. Data are mean±s.d. N=105 nuclei in 3 embryos for TEV injection and 152 nuclei in 3 embryos for buffer injection. Statistical significance calculated by Student's t-test; *P<0.05; ns, not significant. Source data are listed in Table S1.