scholarly journals Organization and composition of apicomplexan kinetochores reveals plasticity in chromosome segregation across parasite modes of division

2021 ◽  
Author(s):  
Lorenzo Brusini ◽  
Nicolas Dos Santos Pacheco ◽  
Dominique Soldati-Favre ◽  
Mathieu Brochet
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Sequence Similarity ◽  
Evolutionary Rates ◽  
Checkpoint Signaling ◽  
Apicomplexan Parasites ◽  
Minimal Structure ◽  
Deep Homology ◽  
Functional Analyses

Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic kinetochore proteins in apicomplexan parasites, in addition to a set of eight apicomplexan components (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm AKiTs are essential for mitosis and reveal architectures parallel to biorientation at metaphase. Furthermore, we identify a homolog of MAD1 at the apicomplexan kinetochore, suggesting conserved spindle assembly checkpoint signaling. Finally, we show unexpected plasticity in kinetochore composition and segregation throughout the parasite lifecycle, indicating diverse requirements to maintain fidelity of chromosome segregation across apicomplexan modes of division.

scholarly journals Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

2016 ◽  
Vol 113 (4) ◽  
pp. 966-971 ◽  
Author(s):  
Sharon Kaisari ◽  
Danielle Sitry-Shevah ◽  
Shirly Miniowitz-Shemtov ◽  
Avram Hershko
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Proteins ◽  
Sister Chromatids ◽  
Unknown Species ◽  
Mitotic Checkpoint Complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

scholarly journals Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

2018 ◽  
Author(s):  
Spyridon T. Pachis ◽  
Yoshitaka Hiruma ◽  
Anastassis Perrakis ◽  
Geert J.P.L. Kops
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Intramolecular Interactions ◽  
Mitotic Progression ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Regulatory Input ◽  
Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

scholarly journals Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ivana Primorac ◽  
John R Weir ◽  
Elena Chiroli ◽  
Fridolin Gross ◽  
Ingrid Hoffmann ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Binding Mode ◽  
Signaling Networks ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Downstream Signaling ◽  
Signaling Components ◽  
Insight Into

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.

scholarly journals Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

2009 ◽  
Vol 184 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Thomas J. Maresca ◽  
Edward D. Salmon
Keyword(s):  
Drosophila Melanogaster ◽  
Green Fluorescent Protein ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Fluorescent Protein ◽  
Spindle Assembly ◽  
Centromeric Chromatin ◽  
Cell Assays ◽  
Green Fluorescent

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.

Driving chromosome segregation: lessons from the human and Drosophila centromere–kinetochore machinery

2010 ◽  
Vol 38 (6) ◽  
pp. 1667-1675 ◽  
Author(s):  
Bernardo Orr ◽  
Olga Afonso ◽  
Tália Feijão ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Genomic Stability ◽  
Mitotic Progression ◽  
Chromosomal Locus ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
And Function ◽  
Drosophila Cells

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

scholarly journals Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

2011 ◽  
Vol 22 (19) ◽  
pp. 3584-3594 ◽  
Author(s):  
Randy Wei ◽  
Bryan Ngo ◽  
Guikai Wu ◽  
Wen-Hwa Lee
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Distribution Characteristic ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Ndc80 Complex ◽  
Complex Protein

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

scholarly journals Molecular mechanism underlying the non-essentiality of Bub1 for the fidelity of chromosome segregation in human cells

2021 ◽  
Author(s):  
Qinfu Chen ◽  
Miao Zhang ◽  
Xuan Pan ◽  
Linli Zhou ◽  
Haiyan Yan ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Chromosome ◽  
Threshold Level ◽  
Underlying Mechanism ◽  
Human Cells ◽  
Strong Reduction ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Redundant Role

SUMMARYThe multi-task protein kinase Bub1 has long been considered important for chromosome alignment and spindle assembly checkpoint signaling during mitosis. However, recent studies provide surprising evidence that Bub1 may not be essential in human cells, with the underlying mechanism unknown. Here we show that Bub1 plays a redundant role with the non-essential CENP-U complex in recruiting Polo-like kinase 1 (Plk1) to the kinetochore. While disrupting either pathway of Plk1 recruitment does not affect the accuracy of whole chromosome segregation, loss of both pathways leads to a strong reduction in the kinetochore accumulation of Plk1 under a threshold level required for proper chromosome alignment and segregation. Thus, parallel recruitment of Plk1 to kinetochores by Bub1 and the CENP-U complex ensures high fidelity of mitotic chromosome segregation. This study may have implications for targeted treatment of cancer cells harboring mutations in either Bub1 or the CENP-U complex.

scholarly journals Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

2018 ◽  
Author(s):  
Cláudia Pereira ◽  
Rita M. Reis ◽  
José B. Gama ◽  
Dhanya K. Cheerambathur ◽  
Ana X. Carvalho ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Self Assembly ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Protein Assembly ◽  
Early Embryo ◽  
Present Evidence ◽  
C Elegans ◽  
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) signalling. 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, while 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 µm-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 RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

scholarly journals Pericentromere tension is self-regulated by spindle structure in metaphase

2014 ◽  
Vol 205 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Jeremy M. Chacón ◽  
Soumya Mukherjee ◽  
Breanna M. Schuster ◽  
Duncan J. Clarke ◽  
Melissa K. Gardner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Chromosome Structure ◽  
Wild Type ◽  
Checkpoint Signaling ◽  
Daughter Cells ◽  
Fluorescent Markers ◽  
Spindle Structure

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4–6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted.

scholarly journals Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Heather E Arsenault ◽  
Julie M Ghizzoni ◽  
Cassandra M Leech ◽  
Anne R Diers ◽  
Stephane Gesta ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Cycle Arrest ◽  
Microtubule Poisons

Abstract The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase-anaphase transition. Here we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging and half-life. Additionally, stabilization of Ubc1 prevented its downregulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that downregulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

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