Abstract
The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.
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Acknowledgements
We thank R. B. Vallee and C. Echeverri for human p50 dynamitin cDNA, R. Saint and W. Sullivan for GFP–histone fly stock, and T. Orr-Weaver for GFP–MeiS332 fly stock. This work was supported by NIH grant no. GM55507 to J.M.S. and NIH postdoctoral fellowship no. GM19262 to D.J.S.
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Movie 1
Control GFP–histone. Time-lapse movie showing chromatid-to-pole movements during anaphase on an individual mitotic spindle from a control-injected embryo. Chromosomes are labelled with GFP–histone (green); spindle microtubules are labelled with rhodamine–tubulin (red). Images were acquired at intervals of ~8.4 s. (MOV 171 kb)
Movie 2
p50 GFP–histone. Time-lapse movie showing chromatid-to-pole movements (equivalent time points to those shown in Movie 1) on an individual spindle from an embryo in which dynein has been inhibited by micro-injection of p50-dynamitin. Chromosomes are labelled with GFP–histone (green); spindle microtubules are labelled with rhodamine–tubulin (red). Note that sister chromatids do not separate but instead decondense at the metaphase plate. (MOV 376 kb)
Movie 3
Control MeiS332. Time-lapse movie (images were acquired at 8.4-s intervals) showing kinetochore-to-pole movements on a single mitotic spindle from a control-injected embryo. Kinetochores are labelled with GFP–MeiS332 (green); spindle microtubules are labelled with rhodamine–tubulin (red). The poleward movement of an individual kinetochore is tracked by an arrow. (MOV 137 kb)
Movie 4
p50 MeiS332. Time-lapse movie showing kinetochore-to-pole movements following the inhibition of dynein by micro-injection of p50 dynamitin. Kinetochores are labelled with GFP–MeiS332 (green); spindle microtubules are labelled with rhodamine–tubulin (red). The movement of an individual kinetochore is tracked by an arrow. Note that under these conditions the kinetochores fail to move polewards from the metaphase plate. (MOV 163 kb)
Movie 5
p50-injection gradient. Time-lapse movie showing the entire surface of a p50-injected embryo and illustrating the gradient of phenotypes observed after injection of dynein inhibitors. The p50-injection site is marked in the first several frames; chromosomes are shown in green and microtubules are shown in red. Note that on nuclei near to the injection site, centrosomes do not completely separate and prophase arrest occurs. Further from the injection site, however, bipolar spindles form but fail to separate their chromosomes. (MOV 5007 kb)
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Sharp, D., Rogers, G. & Scholey, J. Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos. Nat Cell Biol 2, 922–930 (2000). https://doi.org/10.1038/35046574
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DOI: https://doi.org/10.1038/35046574
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