Fig. 6. Spindle checkpoint proteins in anoxia. (A) Selected frames after in vivo imaging of an embryo expressing EGFP-Mps1 (Mps1) and histone H2Av-mRFP (H2Av). Time in minutes is indicated in the frames showing the merged images in the bottom panel. Anoxia (–O₂) was induced at t=1.75 minutes after the onset of chromosome condensation during entry into mitosis. The resulting EGFP-Mps1 accumulation within the spindle midzone region of metaphase figures (arrows) and in centrosome-associated filaments (arrowheads) is indicated. The embryo was reoxygenated (+O₂) at t=9.75 minutes during the metaphase arrest, resulting in EGFP-Mps1 disappearance and completion of mitosis. Bar, 10 µm. (B) Frames showing EGFP-Mps1 at high magnification within the midzone region of a metaphase figure during the anoxia-induced mitotic arrest. Time in seconds is indicated within each frame. An EGFP-Mps1 particle moving along the spindle is indicated by arrowheads. (C) High-magnification views of single nuclei from syncytial gEGFP-Mps1 embryos during interphase. The embryos were fixed and labeled with anti--tubulin (-Tub) and DNA stain (DNA) after a 20-minute incubation in either normoxic (+O₂) or anoxic (–O₂) buffer. The weak centrosomal EGFP-Mps1 (Mps1) signals in normoxic embryos (arrowhead), as well as the strong signals in subcentrosomal dots in anoxic embryos (arrow), are indicated. Bar, 10 µm. (D) EGFP-Mps1 signals (Mps1) in cortical regions of preblastoderm embryos that were fixed after a 20-minute incubation in either normoxic (+O₂) or anoxic (–O₂) buffer. Anoxia induced filamentous or dot-like EGFP-Mps1 aggregates. Bar, 5 µm. (E) gEGFP-BubR1 embryos were fixed and labeled with anti--tubulin (-Tub) and DNA stain (DNA) after a 20-minute incubation in either normoxic (+O₂) or anoxic (–O₂) buffer. In response to anoxia, EGFP-BubR1 (BubR1) accumulates within the midzone region of metaphase figures and in peri-centrosomal filaments (arrowhead). Bar, 5 µm. (F) gEGFP-Mps1 embryos were fixed and labeled with anti-BubR1 (BubR1) and DNA stain (DNA) after a 20-minute incubation in anoxic (–O₂) buffer. EGFP-Mps1 (Mps1) and anti-BubR1 signals largely overlap in the in peri-centrosomal filaments (arrowhead). (G,H,I) gEGFP-Bub3 (G), EGFP-Rod (H) or EGFP-Fzy (I) embryos were fixed after a 20-minute incubation in either normoxic (+O₂) or anoxic (–O₂) buffer and labeled with a DNA stain (G-I; DNA) and anti--tubulin (H; Tub). Bar, 5 µm.