Coordination of NDC80 and Ska complexes at the kinetochore-microtubule interface in human cells

Abstract

The conserved kinetochore-associated NDC80 complex (comprised of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including (1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments, and (2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus-ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochoremicrotubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex, and demonstrate that this region is required for Ska complex recruitment to the NDC80 complexmicrotubule interface. however, is required for high affinity binding of NDC80 complexes to microtubules in vitro (Wei et al., 2007; Ciferri et al., 2008;Miller et al., 2008; Lampert et al., 2013;Umbreit et al., 2012; Alushin et al., 2012; Cheerambathur et al., 2013;Zaytsev et al., 2015), suggesting that cellular factors likely compensate for Hec1 tail domain functions to varying degrees in different organisms.In addition to generating attachments to spindle microtubules, kinetochores also regulate their stability. In early mitosis attachments are labile and undergo rapid turnover, whereas in late mitosis,

Hec1/Ndc80 Tail Domain Function at the Kinetochore-Microtubule Interface

“…Interestingly, stable kinetochore-microtubule attachments formed earlier in these cells compared with cells expressing wild-type Hec1. However, consistent with a role for the tail domain in contributing to force generation at the kinetochore-microtubule interface, inter-kinetochore distances were decreased between sister kinetochores of bi-oriented chromosomes, and cells failed to silence the spindle assembly checkpoint (Wimbish et al, 2019). Based on these phenotypes, we hypothesize that attachments are established prematurely in early mitosis due to an inability to negatively regulate attachments by Aurora kinase-mediated phosphorylation; however, in later mitosis, the tail is required to provide an additional contact point to microtubules to achieve wild-type levels of force generation.…”

“…These results raise the possibility that the Ska complex may be able to, at least in part, compensate for the Hec1 tail domain in human cells. Indeed, a recent study from our lab demonstrated that HeLa cells depleted of Ska complex components and expressing a tail-less Hec1 mutant failed to establish stable kinetochore-microtubule attachments; however, this deficit could be restored when the Ska complex was left unperturbed (Wimbish et al, 2019). Thus, in a manner analogous to the coordinated activities of the NDC80 and Dam1 complexes in budding yeast (Kemmler et al, 2009;Demirel et al, 2012;Suzuki et al, 2016), the Ska complex likely functions in concert with the NDC80 complex in human cells in the establishment and maintenance of stable kinetochoremicrotubule attachments.…”

“…However, recent work from our lab may challenge this notion. In particular, Wimbish et al (2019) demonstrated that the tail is dispensable for the establishment and maintenance of kinetochore-microtubule attachments in HeLa and RPE1 cells. However, in spite of the presence of cold-resistant kinetochoremicrotubule attachments, cells expressing a tail-less Hec1 mutant exhibited defects in chromosome alignment, and experienced long mitotic delays.…”

“…These findings, however, contrast with a number of other studies that examined the Ska complex-NDC80 complex interaction. For example, several groups have reported that the tail domain of human Hec1 is dispensable for Ska complex-mediated enhancement of NDC80 complex-microtubule binding in vitro (Helgeson et al, 2018; Huis in't Veld et al, 2019;Wimbish et al, 2019), and for kinetochore recruitment of Ska complexes in C. elegans and human cells (Cheerambathur et al, 2017;Wimbish et al, 2019). Instead, evidence suggests that the Ska complex contacts the NDC80 complex within the extended coiled-coil domain.…”

“…Instead, evidence suggests that the Ska complex contacts the NDC80 complex within the extended coiled-coil domain. For instance, multiple studies have reported that NDC80 Bonsai complexes, which are missing most of this internal coiledcoil, are unable to interact with Ska complexes (Zhang et al, 2017; Huis in't Veld et al, 2019;Wimbish et al, 2019), presumably because this region mediates the interaction, a notion supported by cross-linking/mass spectrometry data (Helgeson et al, 2018). Thus, although NDC80 complex oligomerization may be part of the mechanism by which Ska complexes enhance NDC80 complex-microtubule binding, this is likely a Hec1 tailindependent phenomenon.…”