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Figure 2


Fig. 2. Mitotic spindle function in anoxia. (A) Spindle checkpoint-deficient embryos lacking Mps1 protein kinase function were incubated during the synchronous syncytial blastoderm cycles in either normoxic (+O2) or anoxic (–O2) buffer for 5 minutes before fixation and labeling with anti-{alpha}-tubulin (Tub) and a DNA stain (DNA). The effects of anoxia on metaphase spindles do not depend on spindle checkpoint function (compare with Fig. 1A). Bar, 10 µm. (B) Progression through mitosis was analyzed by in vivo imaging of Mps1 mutant embryos expressing a green fluorescent Cenp-A/Cid centromere protein (Cenp-A) and a red fluorescent histone H2A variant (Histone H2Av) in either normoxic (+O2, top row) or anoxic (–O2, bottom row) conditions. Time in minutes starting in early prometaphase is indicated in the lower-left corners of the selected frames. Chromosome congression in anoxia is slow and incomplete at the time of anaphase onset. Subsequent chromosome segregation is also slow. Some of the frequent lagging centromeres are indicated by arrowheads. Bar, 10 µm. (C,D) Spindle checkpoint-competent embryos were incubated during the synchronous syncytial blastoderm cycles in either normoxic (+O2) or anoxic (–O2) buffer for 5 minutes before fixation and labeling with antibodies against {alpha}-tubulin (Tub), Cenp-A (Cenp-A) and a DNA stain (DNA). The relative resistance of kinetochore fibers against anoxia-induced depolymerization is illustrated with representative metaphase figures (C). Bar, 5 µm. Moreover, inter-sister kinetochore distances were measured (n=125 sister kinetochore pairs from at least 18 different embryos) and the average (in µm ± s.d.) in prophase (pro) or metaphase (meta) is given in D. The average distance in normoxic (+O2) and anoxic (–O2) metaphase was found to be distinct (P<0.0001 in t-test; n=125). We did not detect significant variation in the average sister kinetochore separation with developmental stage (mitosis 11-13).





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