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

Gama, José B.; Pereira, Cláudia; Simões, Patrícia Alexandra Carvalho; Celestino, Ricardo; Reis, Rita M.; Barbosa, Daniel José; Pires, Helena R.; Carvalho, Cátia A.; Amorim, João Pedro; Carvalho, Alexandra T. P.; Cheerambathur, Dhanya K.; Gassmann, Reto

doi:10.1083/jcb.201610108

Cited by 122 publications

(254 citation statements)

References 71 publications

Supporting

Mentioning

239

Contrasting

Order By: Relevance

“…Mad1 first localized at kinetochores at nuclear envelope breakdown (NEBD), then dissociated as chromosomes congressed at the metaphase plate (Figure 1E and Video S1; n=10 cells). Congression was less efficient in KNTC1 –/– cells, consistent with the lack of Spindly and dynein at kinetochores ([14] and Figure S1F-I), but Mad1 was still targeted to misaligned chromosomes as effectively as in wildtype cells (Figure 1F-G and Video S2; n=14 cells). We conclude that early mitotic cells can recruit Mad1-Mad2 to kinetochores and inhibit anaphase onset in the absence of the RZZ complex.…”

Section: Resultssupporting

confidence: 64%

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

et al. 2018

View full text Add to dashboard Cite

Summary 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 to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.

show abstract

Section: Resultssupporting

confidence: 64%

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Kinetochore expansion is thought to accelerate mitotic 'search and capture' by promoting lateral microtubule attachment [10] and to enhance SAC signaling [11]. RZZ is closely related to endomembrane coatomers that form oligomeric lattices [18,19] and has been suggested as a "building block" of the corona itself [17,20]. Consistently two other corona-associated proteins (CENP-E [21] and CENP-F [22]) also failed to form crescents in KNTC1 -/-cells (Fig.…”

Section: Mps1 Phosphorylates the N-terminus Of Rod To Trigger Kinetocmentioning

confidence: 62%

“…2B; n=10 cells). Congression was less efficient in KNTC-null cells, consistent with the lack of Spindly and dynein at kinetochores ( [17] and Fig. S2), with small numbers of Mad1-positive chromosomes temporarily at the spindle poles ( Fig.…”

Section: Resultsmentioning

confidence: 80%

“…Recent studies indicate that Rod and Spindly not only interact via this domain [17,20] but also inhibit their own self-assembly into polymers [51,52]. In light of these findings and the fact that Mps1 maximally phosphorylates Rod at unattached kinetochores [23], we propose that this modification overcomes the barrier to Spindly-RZZ polymerization and kinetochore expansion.…”

Section: Discussionmentioning

confidence: 99%

“…3D). Thus far the only post-translational modification known to be required for crescent formation is Cterminal farnesylation of Spindly, which enables its kinetochore recruitment via interaction with Rod's beta-propeller domain [17,20,24]. In contrast Rod 2A recruited Spindly normally (Fig.…”

Section: Mps1 Phosphorylates the N-terminus Of Rod To Trigger Kinetocmentioning

confidence: 99%

See 2 more Smart Citations

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

Rodriguez-Rodriguez

McKinley

Sikirzhytski

et al. 2018

Preprint

View full text Add to dashboard Cite

SummaryThe Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls mitosis through assembly of a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1,2]. Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1,2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4][5][6][7][8][9]. Through gene editing and livecell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10][11][12][13] once Mps1 phosphorylates the N-terminus of Rod.Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells. We conclude that RZZ drives structural changes in the outer kinetochore that facilitate chromosome biorientation and chronic SAC transduction, a key determinant of cytotoxicity during antimitotic drug therapy [14][15][16].All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/297580 doi: bioRxiv preprint first posted online Apr. 9, 2018; 3 Results RZZ is required for long-term mitotic arrest in response to spindle poisonsRecent studies have reached different conclusions about the specific roles and relative importance of Bub1 and the RZZ complex in targeting Mad1-Mad2 to kinetochores and transducing SAC arrest [4][5][6][7][8]. To interrogate RZZ function with maximum penetrance, we used AAV (adeno-associated virus)-and CRISPR-mediated gene editing to target the KNTC1 (Rod) locus in HCT116 cells, a diploid colorectal cell line ( Fig. S1A-C). The resulting KNTC1 HF/-(hypomorph-flox) cells expressed Rod at ~20% of the wildtype level ( Fig. 1A-B and S1D) and exited mitosis prematurely when microtubule polymerization (nocodazole, 99 ± 6 min s.e.m.) or Eg5-dependent spindle bipolarity (S-trityl-L-cysteine (STLC), 193 ± 9 min s.e.m.) were inhibited, whereas wildtype cells never exited mitosis during the 16-hour timelapse (Fig. 1A-B). To determine if complete loss of the RZZ complex is compatible with clonogenic survival, KNTC1 HF/-cells were transduced with an adenovirus expressing Cre recombinase (AdCre) and subjected to limiting dilution.Unlike most SAC gene knockouts in mammals, KNTC1 -/-cells were viable (Fig. 1...

show abstract

Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

2019

View full text Add to dashboard Cite

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule‐kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A‐B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1‐cyclin B1 and its counteracting phosphatase PP2A‐B55. Furthermore, we discuss how CDK1‐cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis.

show abstract

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

Abstract: The dynein motor is recruited to the kinetochore to capture spindle microtubules and control the spindle assembly checkpoint. Gama et al. reveal the molecular mechanism of how the Rod–Zw10–Zwilch complex and Spindly mediate dynein recruitment in Caenorhabditis elegans and human cells.

Cited by 122 publications

References 71 publications

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

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

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

Contact Info

Product

Resources

About