The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

Caldas, Gina V.; Lynch, Tina R.; Anderson, Ryan S.; Afreen, Sana; Varma, Dileep; DeLuca, Jennifer G.

doi:10.1098/rsob.150160

Cited by 56 publications

(89 citation statements)

References 30 publications

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“…To test whether over-expression of GFP::ROD promotes kinetochore expansion per se, we expressed GFP::ROD under conditions that usually compromise expansion. Consistent with previous reports [14,17], RNAi-mediated depletion of KNL1 reduced the amount of endogenous RZZ recruited to kinetochores, as revealed by immunostaining for ZWILCH, and kinetochore expansion was significantly suppressed (Fig. 3A).…”

Section: Expression Of Exogenous Rod Promotes Kinetochore Expansionsupporting

confidence: 92%

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Gassmann

Pereira

Reis

et al. 2018

Preprint

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SUMMARYThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into µm-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

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Section: Expression Of Exogenous Rod Promotes Kinetochore Expansionsupporting

confidence: 92%

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Gassmann

Pereira

Reis

et al. 2018

Preprint

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“…It is important to point out that Bub1 only stimulates RZZ recruitment, and if sufficient time is provided, then in the absence of Bub1 the RZZ complex will localize and generate a fibrous corona that can recruit Mad1 (Caldas et al, 2015;Vleugel et al, 2015a). The central Mad1 binding region of Bub1 stimulates RZZ localization, hereby ensuring efficient Mad1 kinetochore localization.…”

Section: Discussionmentioning

confidence: 99%

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

et al. 2019

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Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod‐Zw10‐Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1‐Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1‐Bub1 complex.

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“…This shape change is associated with the motor-dependent release of the Dynein:Dynactin:Spindly:RZZ complex from kinetochores towards spindle poles [318,319,320,321,322,323,324,325]. The central spindle assembly checkpoint component, the Mad1:Mad2 complex, is also removed from kinetochores through this mechanism, effectively terminating SAC signaling by the kinetochore [325,326,327,328,329,330,331]. …”

Section: Linkages Between the Inner And The Outer Kinetochorementioning

confidence: 99%

A Molecular View of Kinetochore Assembly and Function

Musacchio

Desai

2017

Biology

501

589

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Kinetochores are large protein assemblies that connect chromosomes to microtubules of the mitotic and meiotic spindles in order to distribute the replicated genome from a mother cell to its daughters. Kinetochores also control feedback mechanisms responsible for the correction of incorrect microtubule attachments, and for the coordination of chromosome attachment with cell cycle progression. Finally, kinetochores contribute to their own preservation, across generations, at the specific chromosomal loci devoted to host them, the centromeres. They achieve this in most species by exploiting an epigenetic, DNA-sequence-independent mechanism; notable exceptions are budding yeasts where a specific sequence is associated with centromere function. In the last 15 years, extensive progress in the elucidation of the composition of the kinetochore and the identification of various physical and functional modules within its substructure has led to a much deeper molecular understanding of kinetochore organization and the origins of its functional output. Here, we provide a broad summary of this progress, focusing primarily on kinetochores of humans and budding yeast, while highlighting work from other models, and present important unresolved questions for future studies.

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The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

Cited by 56 publications

References 30 publications

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

A Molecular View of Kinetochore Assembly and Function

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