Electron Microscopy.

hTERT RPE cells expressing Centrin1-GFP and CenpA-GFP (50) were fixed for 30 min in PBS containing 2.5% glutaraldehyde (Sigma-Aldrich). EM embedding and serial sectioning were done as previously described (51). We imaged 80-nm sections on a JEM 1400 microscope (JEOL) operated at 80 kV using a side-mounted 4.0-megapixel XR401 sCMOS Camera (AMT). Complete image series recorded at 8K magnification were used to reconstruct partial volumes containing individual midzone bundles. Serial higher-magnification images (25K) were then collected to detail the midzone bundles around chromosomes and microtubule connections between neighboring bundles.