scholarly journals Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

2015 ◽  
Vol 211 (6) ◽  
pp. 1141-1156 ◽  
Author(s):  
Itaru Samejima ◽  
Christos Spanos ◽  
Flavia de Lima Alves ◽  
Tetsuya Hori ◽  
Marinela Perpelescu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Protein Complexes ◽  
Interacting Proteins ◽  
Quantitative Mass Spectrometry ◽  
Mitotic Chromosomes ◽  
Microtubule Binding ◽  
Checkpoint Signaling ◽  
Ndc80 Complex ◽  
Stable Association

Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a “core” kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)–interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding “workhorse” of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore.

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

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 Kinesin-Binding Protein (KBP) buffers the activity of KIF18A and KIF15 in mitosis to ensure accurate chromosome segregation

2018 ◽  
Author(s):  
Heidi L. H. Malaby ◽  
Megan E. Dumas ◽  
Ryoma Ohi ◽  
Jason Stumpff
Keyword(s):  
Motor Activity ◽  
Chromosome Segregation ◽  
Binding Protein ◽  
Mitotic Chromosomes ◽  
Microtubule Binding ◽  
Daughter Cells ◽  
Chromosome Alignment ◽  
Segregation Of Chromosomes ◽  
In Cells

ABSTRACTMitotic kinesins must be regulated to ensure a precise balance of spindle forces and accurate segregation of chromosomes into daughter cells. Here we demonstrate that Kinesin-Binding Protein (KBP) reduces the activity of KIF18A and KIF15 during metaphase. Overexpression of KBP disrupts the movement and alignment of mitotic chromosomes and decreases spindle length, a combination of phenotypes observed in cells deficient for KIF18A and KIF15, respectively. We show through gliding filament and microtubule co-pelleting assays that KBP directly inhibits KIF18A and KIF15 motor activity by preventing microtubule-binding. Consistent with these effects, the mitotic localizations of KIF18A and KIF15 are altered by overexpression of KBP. Cells depleted of KBP exhibit lagging chromosomes in anaphase, an effect that is recapitulated by KIF15 and KIF18A overexpression. Based on these data, we propose a model in which KBP acts as a protein buffer in mitosis, protecting cells from excessive KIF18A and KIF15 activity to promote accurate chromosome segregation.SUMMARYKinesin-Binding Protein (KBP) is identified as a regulator of the kinesins KIF18A and KIF15 during mitosis. KBP buffers the activity of these motors to control chromosome alignment and spindle integrity in metaphase and prevent lagging chromosomes in anaphase.

scholarly journals Prophase removal of chromosome-associated RNAs facilitates anaphase chromosome segregation

2019 ◽  
Author(s):  
Judith A. Sharp ◽  
Wei Wang ◽  
Michael D. Blower
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Nuclear Protein ◽  
Aurora B ◽  
Dna Binding Domain ◽  
Nucleic Acid Binding ◽  
Mitotic Chromosomes ◽  
Nuclear Envelope Breakdown ◽  
Binding Domains

AbstractDuring mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A:RNA complexes on mitotic chromosomes and elevated rates of anaphase segregation defects. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase, and demonstrates that Aurora-B dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

scholarly journals Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
David M Kern ◽  
Julie K Monda ◽  
Kuan-Chung Su ◽  
Elizabeth M Wilson-Kubalek ◽  
Iain M Cheeseman
Keyword(s):  
Chromosome Segregation ◽  
Human Cells ◽  
Cross Linking ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Anaphase Onset ◽  
Ndc80 Complex ◽  
Outstanding Question ◽  
Dynamic Microtubule

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

scholarly journals Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

2012 ◽  
Vol 196 (4) ◽  
pp. 469-482 ◽  
Author(s):  
Julien Espeut ◽  
Dhanya K. Cheerambathur ◽  
Lenno Krenning ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Spindle Checkpoint ◽  
Protein Phosphatase 1 ◽  
Checkpoint Activation ◽  
Load Bearing ◽  
Microtubule Binding ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
N Terminus ◽  
Ndc80 Complex ◽  
Checkpoint Signal

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.

scholarly journals Topoisomerase IIα is essential for maintenance of mitotic chromosome structure

2020 ◽  
Vol 117 (22) ◽  
pp. 12131-12142 ◽  
Author(s):  
Christian F. Nielsen ◽  
Tao Zhang ◽  
Marin Barisic ◽  
Paul Kalitsis ◽  
Damien F. Hudson
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Chromatin Condensation ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Mitotic Chromosome Condensation

Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.

scholarly journals Developmental regulation of mitotic chromosome formation revealed by condensin reporter mice

2020 ◽  
Author(s):  
Gillian C A Taylor ◽  
Lewis A Macdonald ◽  
Matilda Bui ◽  
Lucy Scott ◽  
Ioannis Christodoulou ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Developmental Regulation ◽  
Chromosome Instability ◽  
Cell Lineage ◽  
Mouse Strains ◽  
Mitotic Chromosomes ◽  
Tissue Specific ◽  
Stage Of Development ◽  
Catalytically Active

Germline mutations affecting subunits of condensins I and II cause tissue-specific disease in humans and mice through chromosome segregation failure. However, condensin activity is universally required for chromosome segregation, and hence the developmental basis for these phenotypes is not understood. Using novel transgenic mouse strains, we show that cell-lineage-specific dosage of non-SMC condensin subunits controls the number of catalytically active holocomplexes during different haematopoietic cell divisions in mice. Thymic T cell precursors load significantly higher levels of both condensin I and II subunits onto mitotic chromosomes compared to B cell or erythroid precursors, and undergo elevated mitotic chromosome compaction. Thymic T cells also experience relatively greater chromosome instability in a condensin II hypomorphic strain, indicating that genome propagation requires particularly high condensin activity at this stage of development. Our data highlight developmental changes in the mitotic chromosome condensation pathway, which could contribute to tissue-specific phenotypes in chromosome instability syndromes.

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 Kinesin-binding protein ensures accurate chromosome segregation by buffering KIF18A and KIF15

2019 ◽  
Vol 218 (4) ◽  
pp. 1218-1234 ◽  
Author(s):  
Heidi L.H. Malaby ◽  
Megan E. Dumas ◽  
Ryoma Ohi ◽  
Jason Stumpff
Keyword(s):  
Motor Activity ◽  
Chromosome Segregation ◽  
Binding Protein ◽  
Mitotic Chromosomes ◽  
Microtubule Binding ◽  
Daughter Cells ◽  
Segregation Of Chromosomes ◽  
In Cells

Mitotic kinesins must be regulated to ensure a precise balance of spindle forces and accurate segregation of chromosomes into daughter cells. Here, we demonstrate that kinesin-binding protein (KBP) reduces the activity of KIF18A and KIF15 during metaphase. Overexpression of KBP disrupts the movement and alignment of mitotic chromosomes and decreases spindle length, a combination of phenotypes observed in cells deficient for KIF18A and KIF15, respectively. We show through gliding filament and microtubule co-pelleting assays that KBP directly inhibits KIF18A and KIF15 motor activity by preventing microtubule binding. Consistent with these effects, the mitotic localizations of KIF18A and KIF15 are altered by overexpression of KBP. Cells depleted of KBP exhibit lagging chromosomes in anaphase, an effect that is recapitulated by KIF15 and KIF18A overexpression. Based on these data, we propose a model in which KBP acts as a protein buffer in mitosis, protecting cells from excessive KIF18A and KIF15 activity to promote accurate chromosome segregation.

Sign in / Sign up

Export Citation Format

Share Document