Insights into human outer kinetochore assembly and force transmission from a structure-function analysis of the KMN network

The biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell’s chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. The kinetochore outer layer consists of a 10-subunit apparatus comprising Knl1C, Mis12C, and Ndc80C subcomplexes (KMN network). The KMN network is highly elongated and docks on kinetochores and microtubules using interfaces at its opposite extremes. Here, we combine cryo-EM reconstructions and AlphaFold2 predictions to generate a model of the KMN network that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments and molecular dynamics simulations we demonstrate this mutual organization stabilizes the KMN network. Our work reports the first comprehensive structural and functional analysis of the microtubule binding machinery of kinetochores and elucidates a path of microtubule-generated force transmission