Kara L McKinley1, Nikolina Sekulic2, Lucie Y Guo2, Tonia Tsinman3, Ben E Black2, Iain M Cheeseman4. 1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. 2. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 422 Curie Boulevard, Philadelphia, PA 19104, USA. 3. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA. 4. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: icheese@wi.mit.edu.
Abstract
During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.
During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinpan>etochore assembly are poorly unpan>derstood. Here, we use inpan>ducible CRISPR knpan>ockouts anpan>d biochemical reconpan>stitutionpan>s to definpan>e the inpan>teractionpan>s bepan> class="Chemical">tween the humanCCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.
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