Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

McKinley, Kara L.; Cheeseman, Iain M.

doi:10.1016/j.devcel.2017.01.012

Cited by 181 publications

(215 citation statements)

References 71 publications

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“…Slower NuMA accumulation kinetics after p50 overexpression suggest that dynein-dynactin-NuMA complex formation may aid rapid NuMA recruitment, but dynein 'Walk and Pile-up' alone cannot explain NuMA's minus-end affinity ( Figure 2D,E). To confirm that NuMA localizes to minus-ends even after genetic dynein deletion, we used an inducible CRISPR/Cas9 HeLa cell line to knock out dynein's heavy chain (DHC) (McKinley and Cheeseman, 2017). After dynein knockout (Figure 2-figure supplement 1C,D), both NuMA and dynactin localized robustly to k-fiber minus-ends ( Figure 2F; Figure 2-figure supplement 1E,F).…”

Section: Numa Localizes To Minus-ends Independently Of Dyneinmentioning

confidence: 99%

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Hueschen

Kenny

et al. 2018

Biophysical Journal

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To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA's canonical microtubule-binding domain and is independent of minus-end binders g-TuRC, CAMSAP1, and KANSL1/3. Both NuMA's minus-endbinding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

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Section: Numa Localizes To Minus-ends Independently Of Dyneinmentioning

confidence: 99%

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Hueschen

Kenny

et al. 2018

Biophysical Journal

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“…In contrast, the mutations characterized here disrupt soluble tubulin interactions and microtubule tracking without eliminating binding to the microtubule lattice. To test the effects of these precise mutations on kinetochore function and chromosome segregation, we next utilized an inducible CRISPR/Cas9-based replacement strategy in human HeLa cells [18]. Consistent with prior RNAi-based studies [2, 3, 12, 19–23], induction of the Cas9 nuclease in cells constitutively expressing a guide RNA (sgRNA) targeting Ska1 resulted in severe defects in chromosome alignment and mitotic progression that could be rescued by expression of a guide resistant version of GFP-Ska1 (Figure 4A–D; [18]).…”

Section: Resultsmentioning

confidence: 99%

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

et al. 2017

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Summary The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin, but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation.

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“…We used doxycycline-inducible CRISPR [25] to delete BUB1 in RPE cells with 60% efficiency as judged by IFM, with 42% of cells exiting nocodazole-induced mitotic arrest in less than 800 min ( Fig. S4D-E).…”

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

confidence: 99%

“…Lentiviral transduction was performed as described above. Gene deletion was initiated by inducing a doxycycline-regulated Cas9 transgene present in the host cell line [25] or by infection with AdCas9 (ViraQuest).…”

Section: Aav-mediated Gene Targetingmentioning

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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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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Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

Cited by 181 publications

References 71 publications

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

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

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