Fig. 6: KIF4A-driven sliding is more evident in minimal midzones organized by CDK1-phosphorylated PRC1.

A TIRF microscopy images of microtubule bundles transitioning from an anaphase-to-metaphase-like organization (as in Fig. 4), followed by metaphase-to-anaphase-like transition (as in Fig. 5) using 18 µM Alexa647-tubulin (red), 20 nM PRC1-Alexa546 (green) and 10 nM KIF4A-mGFP (blue). Upon addition of 100 nM CDK1/cyclin B/CKS1, a large fraction of PRC1 and KIF4A detached rapidly from the compacted antiparallel overlaps and the bundles extended, mimicking an anaphase-to-metaphase-like transition. After 10 min, 500 pM λ-PP, 1 mM MnCl₂ and 500 μM CDK1 inhibitor RO-3306 were added to the reservoir to dephosphorylate PRC1 which caused PRC1 and KIF4A to rebind and antiparallel overlaps first extend before they contracted, mimicking a metaphase-to-anaphase transition. Kymograph of TAMRA-tubulin speckles from a microtubule bundle organized at the same condition as in the images above, but using unlabeled PRC1 and additional 0.9 µM of TAMRA-tubulin (bottom). TAMRA-tubulin speckle velocity after CDK1 addition was ~ 1 nm/s initially and later 15 ± 10 nm/s after PRC1 was phosphorylated. n = 2 independent experiments, 36 and 31 speckles, respectively. In contrast, after λ-PP addition the speckle velocity was first 20 ± 9 nm/s, then ~1 nm/s after PRC1 was dephosphorylated. n = 2 independent experiments, 25 and 17 speckles, respectively. B, C TIRF microscopy images showing microtubule bundles with antiparallel overlaps that were pre-organized for 10 min in the presence of (B) 20 nM CDK1-phosphorylated PRC1 or (C) 20 nM of unphosphorylated PRC1 (green) and 18 µM Alexa647-tubulin (red), followed by addition of 30 nM KIF4A-mGFP (blue) to the top reservoir and for delivery to the microscopy chamber by diffusion (top). Kymographs of TAMRA-tubulin speckles generated from TIRF microscopy images of microtubule bundles organized at the same conditions as in images above, but using unlabeled CDK1-phosphorylated PRC1 and additional 0.9 µM TAMRA-tubulin (bottom). TAMRA-tubulin speckle velocity in (B) is 30 ± 10 nm/s and (C) is 0.2 nm/s. n = 2 independent experiments, 42 and 26 speckles, respectively. Times in min:sec, scale bar as indicated. Source data are provided as a Source Data file.