Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint

Abstract

The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD–ZW10–Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1–noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein–hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

Spindly switch controls anaphase: Spindly and RZZ functions in chromosome attachment and mitotic checkpoint control

“…81 Upon establishment of stable end-on and load-bearing Ndc80 complex-mediated microtubule attachments, the kinetochore binding affinity of ZW10 is reduced, which results in a higher dynein-dependent turn-over rate of ZW10 at kinetochores. 81 How this switch is mediated is currently not known but is considered crucial for SAC inactivation. 41,56 Kinetochore stripping commences with stable microtubule attachment and evidence has been obtained that this removal can be asymmetric, affecting only one of sister-kinetochore in the case of monopolar attachments.…”

“…75,80 ZW10 can also be recruited to kinetochores independently of Zwint-1, 81 but, for mitotic checkpoint signaling, a stable Zwint-1-dependent kinetochore interaction of RZZ is essential. 81 Upon establishment of stable end-on and load-bearing Ndc80 complex-mediated microtubule attachments, the kinetochore binding affinity of ZW10 is reduced, which results in a higher dynein-dependent turn-over rate of ZW10 at kinetochores. 81 How this switch is mediated is currently not known but is considered crucial for SAC inactivation.…”

How the SAC gets the axe: Integrating kinetochore microtubule attachments with spindle assembly checkpoint signaling

“…1). [33][34][35][36] However, Zwint1 independent recruitment of the RZZ complex has been reported in multiple studies. 22,28 Earlier, recruitment of the RZZ complex through a direct interaction between Zwint1 and ZW10 of the RZZ complex was proposed to be the mechanism.…”

“…But, normal kinetochore localization of ZW10 mutants defective in Zwint1 binding supported the existence of an alternate mechanism for RZZ recruitment. 35 The surrogate mechanism is accomplished by a central region in BUB1 that could efficiently recruit the RZZ complex to (2106-2316). During prometaphase, when kinetochores are unattached to the microtubules, the SAC is "on."…”

The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing

“…As a negative control, GFP-GW1D1 was coexpressed with GST-tagged fragment N1, the N-terminal amino acids 51-779 of a completely unrelated protein hZW10 (Famulski et al 2008). Unexpectedly, GFP-GW1D1a, GFP-GW1D1b, GFP-GW1D7, and GFP-GW1D5 were all coprecipitated with GST-PIWI (Fig.…”