Fig. 1. Mitotic spindle organization in anoxia. (A) Early Drosophila embryos during the synchronous syncytial blastoderm cycles were incubated in either normoxic (+O₂) or anoxic (–O₂) buffer for 2, 5 or 20 minutes (2', 5', 20') before fixation and labeling with anti--tubulin (Tub) and a DNA stain (DNA). Metaphase embryos reveal an increasing reduction of centrosome-proximal spindle fibers during anoxia. Bar, 10 µm. (B) The intercentrosomal distances in metaphase spindles (in µm on y-axis ± s.d.) after incubation (2 minutes) in either normoxic (white bars) or anoxic (black bars) buffer before fixation and immunolabeling (see A) was measured during mitosis 10, 11, 12 and 13. At least 25 different spindles from at least five different embryos were measured and averaged for each bar (P values obtained with t-test for mitoses 10, 11, 12 and 13 were 0.028, 0.2, 0.035 and 0.77). (C) Selected frames are shown after time-lapse in vivo imaging of embryos expressing -tubulin-GFP. Time in minutes after prometaphase onset is indicated in the lower-left corners. In the presence of oxygen (+O₂, top row), the last metaphase frame is reached after 3.5 minutes, followed by anaphase (not shown). In the embryo made anoxic at the start of mitosis (–O₂, lower row), a metaphase arrest is maintained until after reoxygenation (+O₂, lower row). The last frame before reoxygenation at 15 minutes reveals the loss of centrosome-proximal spindle fibers (arrowheads) and a reduced spindle width (broken lines) in comparison with normoxic metaphase spindles. Reoxygenation is followed by a pronounced increase in centrosomal fibers (18 minutes, arrow) and spindle shortening (21 minutes) before an apparently normal metaphase spindle (27 minutes) precedes anaphase (not shown). (D) The intercentrosomal distance in mitotic spindles was determined after in vivo imaging of embryos expressing GFP-D-TACC starting in prophase. Anaphase onset in normoxic conditions (x-x) is indicated by the open arrow. During the metaphase arrest in anoxic conditions (o-o), the intercentrosomal distance extends slightly beyond normoxic metaphase values. Reoxygenation (indicated by arrowhead +O₂) is followed by a pronounced shortening and eventual anaphase onset (black arrow). The given distances at each time point are average values of five different spindles observed in representative embryos.