Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles, and <i>Caenorhabditiselegans</i> mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles, and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase. Movie S1 Movie S1 Photoconversion (t=0 sec) of microtubules between chromosomes in a wildtype U2OS cell expressing GFP::CENP-A (green) and mEOS3.2::tubulin (green before photoconversion; magenta after photoconversion). Image size, 23 x 23 m. This movie corresponds to Figure 2B. Movie S2 Movie S2 Laser ablation (t=0 sec) of microtubules between chromosomes in a wildtype U2OS cell expressing GFP::CENP-A (green) and mCherry::tubulin (magenta). Image size, 23 x 19 m. This movie corresponds to Figure 3A. Movie S3 Movie S3 Chromosome segregation of a wildtype <i>C. elegans</i> embryo during anaphase. Chromosomes are colored in magenta; microtubules are colored in green. Image size, 48 x 36 m. This movie corresponds to Figure 4A. Movie S4 Movie S4 Chromosome segregation of a <i>
gpr-1/2 (RNAi) C. elegans</i> embryo during anaphase. Chromosomes are colored in magenta; microtubules are colored in green. Image size, 48 x 36 m. This movie corresponds to Figure 4A. Movie S5 Movie S5 Laser ablation (t=0 sec) of microtubules (green) between chromosomes (magenta) in a <i>
gpr-1/2 (RNAi) C. elegans</i> embryo. Image size, 30 x 13 m. This movie corresponds to Figure 5A. Movie S6 Movie S6 Laser ablation (t=18 sec) of microtubules (green) between chromosomes (magenta) and poles (magenta) on both sides of the spindle in a wildtype <i>C. elegans</i> embryo. Image size, 45 x 45 m. This movie corresponds to Figure 5B. Movie S7 Movie S7 Laser ablation (t=0 sec) of microtubules (green) between chromosomes (magenta) in a wildtype <i>C. elegans</i> ooctye in uterus. Image size, 22 x 22 m. This movie corresponds to Figure 6B.