Rod-Zw10-Zwilch Research Articles

The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans

Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments.

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.

ULK1 phosphorylates Mad1 to regulate spindle assembly checkpoint

The spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation during mitosis. Here, we show that ULK1, a serine/threonine kinase that plays a key role in initiation of autophagy, also has an important function in the activation of SAC. ULK1 phosphorylates the SAC protein Mad1 at Ser546 to recruit Mad1 to kinetochores. Furthermore, Rod/ZW10/Zwilch (RZZ) complex may serve as a receptor for phos-Ser546-Mad1 at kinetochore, since phosphorylation of Mad1 by ULK1 strengthens the interaction between Mad1 and RZZ complex. In addition, deletion of ULK1 increases chromosome instability and cytotoxicity of paclitaxel, resulting in significant impairment of cancer cell growth. These findings highlight the role of ULK1 as a protein kinase controlling the fidelity of chromosome segregation and cell-cycle progression.

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

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.

Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

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) signaling. 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, although 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 micrometer-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 reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response tononsense mutations that can reduce or eliminate penetrance in genome editing experiments.

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

Chromosome alignment and segregation during mitosis require kinetochore-microtubule (kMT) attachments that are mediated by the molecular motor dynein and the kMT-binding complex Ndc80. The Rod-ZW10-Zwilch (RZZ) 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 attachments in Caenorhabditis elegans besides its role in activating the spindle assembly checkpoint (SAC). However, the mechanism by which these protein complexes control kMT attachments to drive chromosome motility during early mitosis is still unclear. Here, using in vitro total internal reflection fluorescence microscopy, we observed that higher concentrations of Ndc80 inhibited dynein binding to MTs, providing evidence that Ndc80 and dynein antagonize each other's function. High-resolution microscopy and siRNA-mediated functional disruption revealed that severe defects in chromosome alignment induced by depletion of dynein or the dynein adapter Spindly are rescued by codepletion of the RZZ component Rod in human cells. Interestingly, rescue of the chromosome alignment defects was independent of Rod function in SAC activation and was accompanied by a remarkable restoration of stable kMT attachments. Furthermore, the chromosome alignment rescue depended on the plus-end-directed motility of centromere protein E (CENP-E) because cells codepleted of CENP-E, Rod, and dynein could not establish stable kMT attachments or align their chromosomes properly. Our findings support the idea that dynein may control the function of the Ndc80 complex in stabilizing kMT attachments directly by interfering with Ndc80-MT binding or indirectly by controlling the Rod-mediated inhibition of Ndc80.

The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling.

The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod-ZW10-Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore-microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport-dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.

The RZZ Complex Facilitates Mad1 Binding to Bub1 Ensuring Efficient Checkpoint Signaling

The recruitment of Mad1 to unattached kinetochores is essential for generating a “wait anaphase” signal during mitosis yet Mad1 localization is poorly understood in mammalian cells. In yeast the Bub1 checkpoint protein is the sole Mad1 receptor but in mammalian cells the Rod-ZW10-Zwilch (RZZ) complex is also required for Mad1 kinetochore localization. The exact function of the two mammalian Mad1 receptors and whether there is any interplay between them is unclear. Here we use CRISPR genome editing to generate RNAi sensitized human cell lines revealing a strong requirement for both Rod and Bub1 in checkpoint signaling. We show that the RZZ complex facilitates Mad1 binding to Bub1 and that a region of Bub1 overlapping the Mad1 binding site stimulates RZZ kinetochore recruitment. The requirement for RZZ in the checkpoint, but not Bub1, can be bypassed by tethering Mad1 to kinetochores or by increasing the strength of the Bub1-Mad1 interaction. Our data support a model in which the primary role of RZZ is to localize Mad1 at kinetochores allowing for the efficient checkpoint generating Mad1-Bub1 interaction. As such, the core checkpoint principle is conserved from yeast to man.

Dynein at kinetochores: Making the connection.

Dynein removes the checkpoint proteins from kinetochores once chromosomes are bioriented. In this issue, Gama et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201610108) and Mosalaganti et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611060) reveal the molecular basis for how dynein and its adaptor protein Spindly are recruited to the ROD-Zw10-Zwilch complex in the fibrous corona of unattached kinetochores.

Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly.

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod-Zw10-Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod's N-terminal β-propeller and the associated Zwilch subunit bind Spindly's C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod β-propeller-Zwilch complex in vitro. Spindly's N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein-dynactin-Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein-dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores

The spindle assembly checkpoint (SAC) ensures the accurate segregation of sister chromatids during mitosis. Activation of the SAC occurs through a series of ordered molecular events that result in recruitment of Mad1:Mad2 complexes to improperly attached kinetochores. The current model involves sequential phospho-dependent recruitment of Bub3:Bub1 to KNL1 followed by binding of Mad1:Mad2 to Bub1. Here, we show in non-transformed diploid human cells that the KNL1-Bub3-Bub1 (KBB) pathway is required during normal mitotic progression when kinetochores are misaligned but is nonessential for SAC activation and Mad2 loading when kinetochores are unattached from microtubules. We provide evidence that the Rod-ZW10-Zwilch (RZZ) complex is necessary to recruit Mad1:Mad2 to, and delay anaphase onset in response to, unattached kinetochores independently of the KBB pathway. These data suggest that the KBB and RZZ complexes provide two distinct kinetochore receptors for Mad1:Mad2 and reveal mechanistic differences between SAC activation by unattached and improperly attached kinetochores.

A maternal effect rough deal mutation suggests that multiple pathways regulate Drosophila RZZ kinetochore recruitment.

Proper kinetochore recruitment and regulation of dynein and the Mad1-Mad2 complex requires the Rod-Zw10-Zwilch (RZZ) complex. Here, we describe rod(Z3), a maternal-effect Drosophila mutation changing a single residue in the Rough Deal (Rod) subunit of RZZ. Although the RZZ complex containing this altered subunit (denoted R(Z3)ZZ) is present in early syncytial stage embryos laid by homozygous rod(Z3) mothers, it is not recruited to kinetochores. Consequently, the embryos have no spindle assembly checkpoint (SAC), and syncytial mitoses are profoundly perturbed. The polar body (residual meiotic products) cannot remain in its SAC-dependent metaphase-like state, and decondenses into chromatin. In neuroblasts of homozygous rod(Z3) larvae, R(Z3)ZZ recruitment is only partially reduced, the SAC is functional and mitosis is relatively normal. R(Z3)ZZ nevertheless behaves abnormally: it does not further accumulate on kinetochores when microtubules are depolymerized; it reduces the rate of Mad1 recruitment; and it dominantly interferes with the dynein-mediated streaming of RZZ from attached kinetochores. These results suggest that the mutated residue of rod(Z3) is required for normal RZZ kinetochore recruitment and function and, moreover, that the RZZ recruitment pathway might differ in syncytial stage embryos and post-embryonic somatic cells.

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

RZZ Finds Its Ancestral Roots

In this issue of Structure, Civril et al. (2010) describe an unexpected structural similarity between the kinetochore component Rod-Zw10-Zwilch (RZZ) and the vesicle transport component Nag-Rint1-Zw10 (NRZ).

Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle-regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NDelta253), or the conserved Spindly box (DeltaSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly

Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD–ZW10–ZWILCH complex. In two-cell–stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

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

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.