Highlights d Lepidopteran kinetochores lack CenH3 but contain CCAN homologs essential for mitosis d CENP-I depletion impairs Mis12 and Ndc80 complex recruitment d CENP-T is sufficient to recruit the Ndc80 and Mis12 complexes d CENP-T and other CCANs are present in independently derived CenH3-deficient insects
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromereassociated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPRmediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a 2 conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.
IntroductionThe centromere is an essential chromosomal region which ensures equal partitioning of chromosomal DNA during cell division [1]. In all eukaryotes, faithful chromosome segregation requires each chromosome to interact accurately with microtubule fibers from the mitotic or meiotic spindle. This interaction is mediated by the kinetochore, a macromolecular protein complex that assembles on centromeric DNA [2]. The centromere-proximal inner kinetochore hosts components of the Constitutive Centromere Associated Network (CCAN), a group of up to 16 different proteins present throughout the cell cycle that create the centromere-kinetochore interface. Upon cell division, the CCAN recruits the centromere distal outer kinetochore complex, which is composed of the KMN network (Knl1, the Mis12 and the Ndc80 complex) [3]. This recruitment is enabled by two CCAN subunits, CENP-C and CENP-T, that physically interact with subunits of the Mis12 and Ndc80 complexes [4][5][6][7][8][9][10][11]. The KMN network then in turn mediates the interaction with spindle microtubules to drive chromosome segregation during cell division [12].Previous work identified the histone H3 variant CenH3 (also called CENP-A [13,14]) as a core constituent for defining the site of a functional kinetochore in most eukaryotes. This is because CenH3 forms specialized nucleosomes preferentially found at centromeres that are the target sites for kinetochore assembly [15][16][17][18]. To do so, CenH3 physically in...
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