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

Rodriguez-Rodriguez, José-Antonio; McKinley, Kara L.; Sikirzhytski, Vitali; Corona, Jennifer; Maciejowski, John; Khodjakov, Alexey; Cheeseman, Iain M.; Jallepalli, Prasad V.

doi:10.1101/297580

Cited by 5 publications

(8 citation statements)

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“…This is exemplified by the outermost “fibrous corona” in human cells, which is an expanded region that forms around kinetochores during prometaphase to aid the capture of microtubules (Cassimeris et al, 1990 ; Thrower et al, 1996 ; Hoffman et al, 2001 ; Magidson et al, 2015 ; Wynne and Funabiki, 2015 ). Integral to the structure of the corona is the Rod/Zwilch/ZW10 (RZZ) complex, which forms oligomers that drive rapid corona expansion (Gama et al, 2017 ; Mosalaganti et al, 2017 ; Gassmann et al, 2018 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ). The RZZ complex also helps to engage the SAC and many different SAC proteins localize to the expanded corona (Basto et al, 2000 ; Buffin et al, 2005 ; Kops et al, 2005 ; Silió et al, 2015 ; Wynne and Funabiki, 2015 ; Gassmann et al, 2018 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

“…Integral to the structure of the corona is the Rod/Zwilch/ZW10 (RZZ) complex, which forms oligomers that drive rapid corona expansion (Gama et al, 2017 ; Mosalaganti et al, 2017 ; Gassmann et al, 2018 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ). The RZZ complex also helps to engage the SAC and many different SAC proteins localize to the expanded corona (Basto et al, 2000 ; Buffin et al, 2005 ; Kops et al, 2005 ; Silió et al, 2015 ; Wynne and Funabiki, 2015 ; Gassmann et al, 2018 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ). In fact, the ability of the corona to support SAC signaling may help to explain the puzzling recent observations that Bub1 is dispensable for the SAC in human cells (Currie et al, 2018 ; Raaijmakers et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

“…It will be important to understand how the corona collaborates with the KMN network to regulate chromosome segregation, and in particular, how the enzymes at the KMN network may drive corona assembly and disassembly. Very recent work has begun to shed light on this interplay (Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

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Kinase and Phosphatase Cross-Talk at the Kinetochore

Saurin

2018

Front. Cell Dev. Biol.

113

125

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Multiple kinases and phosphatases act on the kinetochore to control chromosome segregation: Aurora B, Mps1, Bub1, Plk1, Cdk1, PP1, and PP2A-B56, have all been shown to regulate both kinetochore-microtubule attachments and the spindle assembly checkpoint. Given that so many kinases and phosphatases converge onto two key mitotic processes, it is perhaps not surprising to learn that they are, quite literally, entangled in cross-talk. Inhibition of any one of these enzymes produces secondary effects on all the others, which results in a complicated picture that is very difficult to interpret. This review aims to clarify this picture by first collating the direct effects of each enzyme into one overarching schematic of regulation at the Knl1/Mis12/Ndc80 (KMN) network (a major signaling hub at the outer kinetochore). This schematic will then be used to discuss the implications of the cross-talk that connects these enzymes; both in terms of why it may be needed to produce the right type of kinetochore signals and why it nevertheless complicates our interpretations about which enzymes control what processes. Finally, some general experimental approaches will be discussed that could help to characterize kinetochore signaling by dissociating the direct from indirect effect of kinase or phosphatase inhibition in vivo. Together, this review should provide a framework to help understand how a network of kinases and phosphatases cooperate to regulate two key mitotic processes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Kinase and Phosphatase Cross-Talk at the Kinetochore

Saurin

2018

Front. Cell Dev. Biol.

113

125

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“…The ability of Bub1 and RZZ to independently generate a weak checkpoint signal likely explains why mammalian cells that efficiently align their chromosomes can propagate without one of the Mad1 receptors although adaption in these cell lines cannot be presently excluded 20,30,31 . The combination of CRISPR and RNAi used here avoids issues with adaption while still obtaining penetrant depletion and robust phenotypes that can be rescued with RNAi resistant constructs.…”

Section: Main Textmentioning

confidence: 99%

The RZZ complex facilitates Mad1 binding to Bub1 ensuring efficient checkpoint signaling

Zhang

Kruse

Nilsson

2018

Preprint

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Introductory paragraphThe 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.. CC-BY-NC-ND 4.0 International license It is made available under a (which 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 . http://dx.doi.org/10.1101/322834 doi: bioRxiv preprint first posted online May. 15, 2018; 2 Main textIn response to unattached kinetochores the spindle assembly checkpoint (SAC) is activated resulting in kinetochore recruitment of Mad1/Mad2, Bub1 and BubR1 checkpoint proteins in a manner stimulated by Mps1 kinase activity 1 . The recruitment of the Mad1/Mad2 complex, mediated by Mad1 kinetochore interactions, is essential because it catalyzes the first step in the generation of the mitotic checkpoint complex (MCC) that is the SAC effector delaying anaphase onset 2-5 .Understanding Mad1 interactions at kinetochores is therefore crucial for understanding the mechanism of SAC activation. In yeast Bub1 is the only receptor for Mad1 and formation of the Mad1-Bub1 complex requires Mps1 phosphorylation of conserved domain 1 (CD1) in Bub1 6-8 . This mode of Mad1-Bub1 interaction is conserved to mammalian cells 4,5,9,10 . However in mammalian cells the RZZ complex also contributes to localization of Mad1 and checkpoint activity (Supplemental Fig. 1) [11][12][13][14][15] . Several conflicting models regarding the specific mechanistic role and relative importance of the two Mad1 receptors in SAC signaling have been proposed 10,[16][17][18][19][20] . This is possibly due to varying degrees of depletion efficiencies and the fact that even minute amounts of checkpoint proteins are able to generate a checkpoint signal 21 . Furthermore whether there is any interplay between the two Mad1 receptors remains unexplored. Here we find a strong requirement for both Rod and Bub1 in generating a checkpoint signal a...

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“…In these poorly-understood processes, the presence of a nonsense mutation causes cells to produce an alternative transcript that bypasses the CRISPR-induced mutation. While the resulting transcripts may differ from the dominant isoform expressed in a cell line, these transcripts can still be sufficient to carry out the function of the protein (Mou et al, 2017;Rodriguez-Rodriguez et al, 2018). Secondly, while CRISPR-mediated mutagenesis can effectively ablate the targeted locus, the resulting indel can itself have unintended consequences.…”

Section: Introductionmentioning

confidence: 99%

Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

Giuliano

Lin

Sheltzer

2019

Preprint

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CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.

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

Cited by 5 publications

References 54 publications

Kinase and Phosphatase Cross-Talk at the Kinetochore

Kinase and Phosphatase Cross-Talk at the Kinetochore

The RZZ complex facilitates Mad1 binding to Bub1 ensuring efficient checkpoint signaling

Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

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