Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad1-Dependent Anaphase Inhibitor

Rodríguez-Bravo, Verónica; Maciejowski, John; Corona, Jennifer; Buch, Håkon Kirkeby; Collin, Philippe; Kanemaki, Masato T.; Shah, Jagesh V.; Jallepalli, Prasad V.

doi:10.1016/j.cell.2014.01.010

Cited by 157 publications

(247 citation statements)

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“…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].…”

Section: Resultsmentioning

confidence: 99%

“…When both steps are abrogated, cells neither initiate nor maintain the SAC and thus exit mitosis with kinetics set by nuclear pore-derived MCC [3].…”

Section: Discussionmentioning

confidence: 99%

“…This contribution was revealed when cells bearing an NPC-defective Mad1 allele were treated with Aurora B inhibitors, which delay kinetochore recruitment of Mad1 relative to NEBD [31,32]. Under these conditions, cells entered mitosis without premade MCC and could not generate it quickly enough at kinetochores to prevent mitotic exit [3]. Because RZZ-deficient cells initiate but do not maintain the SAC, we anticipated a similar synthetic interaction with Aurora B inhibition.…”

Section: Aurora B and Rzz Target Mad1-mad2 To Kinetochores And Promotmentioning

confidence: 99%

“…NPC-bound Mad1-Mad2 generates MCC during interphase, thereby increasing the SAC's sensitivity and robustness [3]. This contribution was revealed when cells bearing an NPC-defective Mad1 allele were treated with Aurora B inhibitors, which delay kinetochore recruitment of Mad1 relative to NEBD [31,32].…”

Section: Aurora B and Rzz Target Mad1-mad2 To Kinetochores And Promotmentioning

confidence: 99%

“…The first occurs at NEBD, when Mad1-Mad2 released from nuclear pores is targeted to newly assembled kinetochores by Aurora B [3,31,43]. In the second step, Mad1-Mad2 becomes stably tethered to kinetochores by RZZ, extending SAC surveillance and mitotic arrest more than 10-fold.When both steps are abrogated, cells neither initiate nor maintain the SAC and thus exit mitosis with kinetics set by nuclear pore-derived MCC [3].Compelling evidence indicates that Bub1 is the kinetochore receptor for Mad1-Mad2 in yeast and nematodes [44,45]. In contrast, studies in mammalian cells differ as to (1) whether Bub1 recruits Mad1-Mad2 to kinetochores and (2) whether and how Bub1 contributes to SAC arrest [4-7, 9, 41].…”

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

Rodriguez-Rodriguez

McKinley

Sikirzhytski

et al. 2018

Preprint

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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...

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Section: Resultsmentioning

confidence: 99%

“…When both steps are abrogated, cells neither initiate nor maintain the SAC and thus exit mitosis with kinetics set by nuclear pore-derived MCC [3].…”

Section: Discussionmentioning

confidence: 99%

Section: Aurora B and Rzz Target Mad1-mad2 To Kinetochores And Promotmentioning

confidence: 99%

Section: Aurora B and Rzz Target Mad1-mad2 To Kinetochores And Promotmentioning

confidence: 99%

mentioning

confidence: 99%

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

Rodriguez-Rodriguez

McKinley

Sikirzhytski

et al. 2018

Preprint

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From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability

Cunha-Silva

Conde

2020

BioEssays

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The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1-c-Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1-c-Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate-limiting step in the assembly of anaphase-inhibitory complexes. SAC proficiency further requires Mad1-c-Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill-defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1-c-Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1-c-Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted.

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Prostate cancer research: The next generation; report from the 2019 Coffey‐Holden Prostate Cancer Academy Meeting

et al. 2019

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Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad1-Dependent Anaphase Inhibitor

Cited by 157 publications

References 90 publications

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

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

From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability

Prostate cancer research: The next generation; report from the 2019 Coffey‐Holden Prostate Cancer Academy Meeting

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