CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T

Abstract

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

The structural basis for kinetochore stabilization by Cnn1/CENP-T

“…Restriction of Ctf19c-independent Cnn1 localization to anaphase suggests any such contribution must be Cdk1-independent. It remains to be seen how this regulatory logic has been modified in organisms that rely solely on the Cnn1/CENP-T pathway for kinetochoremicrotubule contact (Cortes- Silva et al, 2020).…”

The molecular architecture of CenH3-deficient holocentromeres in Lepidoptera is dependent on transcriptional and chromatin dynamics

“…Given that CenH3 in insects is essential in monocentric Diptera (Blower and Karpen, 2001) and its loss is only found in holocentric lineages (Drinnenberg et al, 2014), the establishment of this derived holocentric state is likely to have preceded and subsequently allowed the loss of CenH3. This is also supported by the fact that some holocentric Hemipteran insects have CenH3 homologs (Cortes-Silva et al, 2020). These Hemipterans could therefore represent an intermediate form leading to the establishment of the CenH3-deficient state.…”

“…While CENP-T is an inner kinetochore component that binds directly to DNA as shown in vertebrates (Hori et al, 2008), the centromere-distal outer kinetochore components bridge centromeric DNA to the spindle apparatus, thus serving as a proxy for potential sites of spindle fiber attachment (Musacchio and Desai, 2017). To test whether the broad localization pattern we observed for CENP-T corresponds also to sites of outer kinetochore assembly, we co-stained our chromosome spreads with another custom-made antibody specific for the outer kinetochore component Dsn1 (Cortes-Silva et al, 2020). We found that the CENP-T and Dsn1 immunosignals co-localize with one another ( Figure 1B), demonstrating that diffuse regions along the poleward surface of B. mori sister chromatids represent potential sites driving chromosome segregation during mitosis.…”

“…To understand holocentromere architecture in CenH3-deficient insects, we used a cell line derived from our representative insect model system, B. mori. We targeted one previously identified centromereproximal component of the B. mori kinetochore, CENP-T (Cortes-Silva et al, 2020) to visualize its localization pattern on mitotic B. mori chromosome spreads by immunofluorescence (IF) microscopy using a custom-made antibody (Cortes-Silva et al, 2020). We observed that the CENP-T-specific IF signal formed broad localization patterns along the polar length of sister chromatids ( Figure 1A), a pattern reminiscent to kinetochore staining in other holocentric organisms (Buchwitz et al, 1999).…”

“…This pattern was highly reproducible across replicates ( Figure S1A and S1B). Given that the ChIP-seq libraries were prepared from asynchronous cell populations containing ~ 2% mitotic cells (Cortes-Silva et al, 2020) and that the B. mori CENP-T, as the vertebrate CENP-T homologs (Hori et al, 2008) also localizes to chromatin during interphase ( Figure S1E), CENP-T interphasic localization likely contributes to the majority of its enrichment signal.…”