Chromokinesin KLP-19 regulates microtubule overlap and dynamics during anaphase in C. elegans

Recent studies have highlighted the significance of the spindle midzone in ensuring proper chromosome segregation. Using 3D electron tomography, light microscopy and a single-cell in vitro assay for single-molecule tracking, we investigated the role of the C. elegans KIF4A homolog KLP-19 in the spindle midzone. Using second harmonic generation and two-photon fluorescence microscopy, we provide the first in vivo measurement of microtubule polarity in the anaphase spindle midzone, showing that the antiparallel microtubule overlap length remains constant and is independent of cortical pulling forces and the bundling protein SPD-1 (the homolog of PRC1). Instead, KLP-19 actively regulates overlap length by controlling microtubule dynamics within the midzone. KLP-19 depletion increases microtubule length, overlap and interaction, correlating with reduced rates of pole and chromosome separation. Using a single-cell in vitro assay, we show KLP-19 is a dimeric processive motor. Yeast-2-hybrid assays confirm a direct KLP-19-SPD-1 interaction, yet KLP-19 midzone localization does not strictly require SPD-1. Together, our data establish KLP-19 as a key regulator of midzone microtubule dynamics required for efficient chromosome segregation.