Antagonism between the dynein and Ndc80 complexes at kinetochores controls the stability of kinetochore-microtubule attachments during mitosis

Abstract

Chromosome alignment and segregation during mitosis depends critically on kinetochoremicrotubule (kMT) attachments that are mediated by the function of the molecular motor cytoplasmic dynein, and the kinetochore microtubule (MT) binding complex, Ndc80. The RZZ (Rod-ZW10-Zwilch) complex is central to this coordination as it has an important role in dynein recruitment and has recently been reported to have a key function in the regulation of stable kMT attachment formation in C. elegans. However, the mechanism by which kMT attachments are controlled by the coordinated function of these protein complexes to drive chromosome motility during early mitosis is still unclear. In this manuscript, we provide evidence to show that Ndc80 and dynein directly antagonize each other's MT-binding. We also find that severe chromosome alignment defects induced by depletion of dynein, or the dynein adapter spindly, are rescued by codepletion of the RZZ component, Rod, in human cells. Interestingly, the rescue of chromosome alignments defects was independent of Rod function in activation of the spindle assembly checkpoint and was accompanied by a remarkable restoration of stable kMT attachments.Furthermore, rescue of chromosome alignment was critically dependent on the plus-end-directed motility of CENP-E, as cells codepleted of CENP-E along with Rod and dynein were unable to establish stable kMT attachments or align their chromosomes properly. Taken together, our findings support the idea that the dynein motor may control the function of the Ndc80 complex in stabilizing kMT attachments either directly by interfering with Ndc80-MT binding, and/or indirectly by modulating the Rod-mediated inhibition of Ndc80.peer-reviewed)

The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

“…We first confirmed the localization to these proteins to prometaphase kinetochores during early mitosis (Fig S2A). Next, we disrupted their function by treating with specific siRNAs and employing published protocols (Amin et al, 2015; Amin et al, 2018; Kiyomitsu et al, 2007). We confirmed the efficiency of depletion of the target proteins by immunoblotting (Fig S2B).…”

“…It has been previously shown that chromosomes captured by spindle microtubules move poleward rapidly, via lateral kMT attachments and motility mediated by dynein (Rieder and Alexander, 1990; Vorozhko et al, 2008; Yang et al, 2007). In vitro analysis showed that microtubule-binding to kinetochores is reduced by dynein/dynactin inhibition (Vorozhko et al, 2008), and that kinetochore dynein prevents premature stabilization of load-bearling, end-on kMT attachments (Amin et al, 2018; Barisic and Maiato, 2015; Cheerambathur et al, 2013; Gassmann et al, 2008). Our data suggest that dynein, however, is not required for a large fraction of initial kMT attachments that are formed during early mitosis in Human cells (Fig S2C, D).…”

“…Recent studies have reported that the Ndc80-mediated stabilization of load-bearing kMT attachments prematurely during early mitosis is inhibited by kinetochore dynein (Amin et al, 2018; Cheerambathur et al, 2013; Gassmann et al, 2008). From our current findings we surmise that the function of Ndc80 complex in forming initial kMT attachments during early mitosis should precede the onset of inhibition of its function in kMT stabilization by dynein.…”

“…siRNAs targeting 5’ UTR Ndc80 (DeLuca et al, 2011), 3’ UTR Dynein (Amin et al, 2018), and siRNAs targeting cDNAs for CLIP-170 and CENP-E (Amin et al, 2015), Nuf2 (DeLuca et al, 2002), Knl1 (Kiyomitsu et al, 2007) were used this study. The Smart Pool ON-TARGET plus siRNAs was used for Spindly knockdown (Amin et al, 2018). All siRNAs were used at 100 nM concentration except for dynein and CLIP-170 (200 nM).…”

“…It has previously been shown that Aurora B kinase activity is required to recruit the microtubule motors, dynein and CENP-E that are responsible for chromosome congression to the kinetochores (Ditchfield et al, 2003;Kasuboski et al, 2011;Murata-Hori et al, 2002). Moreover, our previous findings showed that Ndc80 competes with dynein to bind microtubules (Amin et al, 2018). We first tested if there is a physical association of Ndc80 with the two kinetochore microtubule motors in early mitosis by an immunoprecipitation assay.…”