scholarly journals Elevated polar ejection forces stabilize kinetochore–microtubule attachments

2013 ◽  
Vol 200 (2) ◽  
pp. 203-218 ◽  
Author(s):  
Stuart Cane ◽  
Anna A. Ye ◽  
Sasha J. Luks-Morgan ◽  
Thomas J. Maresca
Keyword(s):  
Molecular Motors ◽  
Molecular Mechanisms ◽  
Critical Force ◽  
Aurora B ◽  
Dependent Manner ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Alignment ◽  
Directed Motility ◽  
Spindle Microtubules

Chromosome biorientation promotes congression and generates tension that stabilizes kinetochore–microtubule (kt-MT) interactions. Forces produced by molecular motors also contribute to chromosome alignment, but their impact on kt-MT attachment stability is unclear. A critical force that acts on chromosomes is the kinesin-10–dependent polar ejection force (PEF). PEFs are proposed to facilitate congression by pushing chromosomes away from spindle poles, although knowledge of the molecular mechanisms underpinning PEF generation is incomplete. Here, we describe a live-cell PEF assay in which tension was applied to chromosomes by manipulating levels of the chromokinesin NOD (no distributive disjunction; Drosophila melanogaster kinesin-10). NOD stabilized syntelic kt-MT attachments in a dose- and motor-dependent manner by overwhelming the ability of Aurora B to mediate error correction. NOD-coated chromatin stretched away from the pole via lateral and end-on interactions with microtubules, and NOD chimeras with either plus end–directed motility or tip-tracking activity produced PEFs. Thus, kt-MT attachment stability is modulated by PEFs, which can be generated by distinct force-producing interactions between chromosomes and dynamic spindle microtubules.

scholarly journals Molecular mechanisms for microtubule length regulation by kinesin-8 and XMAP215 proteins

2014 ◽  
Vol 4 (6) ◽  
pp. 20140031 ◽  
Author(s):  
Louis Reese ◽  
Anna Melbinger ◽  
Erwin Frey
Keyword(s):  
Molecular Motors ◽  
Molecular Mechanisms ◽  
Molecular Motor ◽  
Mutual Exclusion ◽  
High Growth ◽  
Dependent Manner ◽  
Dynamic Regulation ◽  
Filament Dynamics ◽  
Length Regulation ◽  
The Stability

The cytoskeleton is regulated by a plethora of enzymes that influence the stability and dynamics of cytoskeletal filaments. How microtubules (MTs) are controlled is of particular importance for mitosis, during which dynamic MTs are responsible for proper segregation of chromosomes. Molecular motors of the kinesin-8 protein family have been shown to depolymerize MTs in a length-dependent manner, and recent experimental and theoretical evidence suggests a possible role for kinesin-8 in the dynamic regulation of MTs. However, so far the detailed molecular mechanisms of how these molecular motors interact with the growing MT tip remain elusive. Here we show that two distinct scenarios for the interactions of kinesin-8 with the MT tip lead to qualitatively different MT dynamics, including accurate length control as well as intermittent dynamics. We give a comprehensive analysis of the regimes where length regulation is possible and characterize how the stationary length depends on the biochemical rates and the bulk concentrations of the various proteins. For a neutral scenario, where MTs grow irrespective of whether the MT tip is occupied by a molecular motor, length regulation is possible only for a narrow range of biochemical rates, and, in particular, limited to small polymerization rates. By contrast, for an inhibition scenario, where the presence of a motor at the MT tip inhibits MT growth, the regime where length regulation is possible is extremely broad and includes high growth rates. These results also apply to situations where a polymerizing enzyme like XMAP215 and kinesin-8 mutually exclude each other from the MT tip. Moreover, we characterize the differences in the stochastic length dynamics between the two scenarios. While for the neutral scenario length is tightly controlled, length dynamics is intermittent for the inhibition scenario and exhibits extended periods of MT growth and shrinkage. On a broader perspective, the set of models established in this work quite generally suggest that mutual exclusion of molecules at the ends of cytoskeletal filaments is an important factor for filament dynamics and regulation.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

scholarly journals EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

2014 ◽  
Vol 204 (6) ◽  
pp. 947-963 ◽  
Author(s):  
Budhaditya Banerjee ◽  
Cortney A. Kestner ◽  
P. Todd Stukenberg
Keyword(s):  
Mitotic Chromosome ◽  
Histone H3 ◽  
Aurora B ◽  
Dependent Manner ◽  
Histone H2a ◽  
Aurora B Kinase ◽  
Chromosomal Passenger Complex ◽  
Checkpoint Signaling ◽  
Chromosomal Passenger ◽  
Spindle Microtubules

The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.

scholarly journals SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation

2021 ◽  
Author(s):  
Manjuan Zhang ◽  
Fengrui Yang ◽  
Wenwen Wang ◽  
Xiwei Wang ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Spindle Microtubule ◽  
Dynamic Interactions ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
And Behavior

Abstract Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

scholarly journals Aurora B kinase controls the targeting of the Astrin–SKAP complex to bioriented kinetochores

2010 ◽  
Vol 191 (2) ◽  
pp. 269-280 ◽  
Author(s):  
Jens C. Schmidt ◽  
Tomomi Kiyomitsu ◽  
Tetsuya Hori ◽  
Chelsea B. Backer ◽  
Tatsuo Fukagawa ◽  
...  
Keyword(s):  
Error Correction ◽  
Spindle Assembly Checkpoint ◽  
Multiple Roles ◽  
Aurora B ◽  
Dependent Manner ◽  
Dynein Light Chain ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Mitotic Delay ◽  
Associated Proteins

During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B–dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin–SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint–dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.

scholarly journals The chromosomal passenger complex: guiding Aurora-B through mitosis

2006 ◽  
Vol 173 (6) ◽  
pp. 833-837 ◽  
Author(s):  
Gerben Vader ◽  
René H. Medema ◽  
Susanne M.A. Lens
Keyword(s):  
Cell Division ◽  
Histone Modification ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Aurora B ◽  
Chromosomal Passenger Complex ◽  
Precise Control ◽  
Chromosome Alignment ◽  
Chromosomal Passenger ◽  
And Function

During mitosis, the chromosomal passenger complex (CPC) orchestrates highly different processes, such as chromosome alignment, histone modification, and cytokinesis. Proper and timely localization of this complex is the key to precise control over the enzymatic core of the CPC, the Aurora-B kinase. We discuss the molecular mechanisms by which the CPC members direct the dynamic localization of the complex throughout cell division. Also, we summarize posttranslational modifications that occur on the CPC and discuss their roles in regulating localization and function of this mitotic complex.

scholarly journals Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

2015 ◽  
Vol 208 (2) ◽  
pp. 181-196 ◽  
Author(s):  
Soonjoung Kim ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Aurora B ◽  
Dependent Manner ◽  
Checkpoint Proteins ◽  
Multiple Strategies ◽  
Complete Inactivation ◽  
Microtubule Attachment ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Multiple Assembly

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

scholarly journals The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

2019 ◽  
Author(s):  
Luciano Gama Braga ◽  
Angel F. Cisneros ◽  
Michelle Mathieu ◽  
Maxime Clerc ◽  
Pauline Garcia ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Aurora B ◽  
Dependent Manner ◽  
Regulatory Domain ◽  
Checkpoint Protein ◽  
Major Player ◽  
Chromosome Alignment ◽  
Spindle Assembly Checkpoint Protein

ABSTRACTThe balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

scholarly journals Structural basis of human kinesin-8 function and inhibition

2017 ◽  
Vol 114 (45) ◽  
pp. E9539-E9548 ◽  
Author(s):  
Julia Locke ◽  
Agnel Praveen Joseph ◽  
Alejandro Peña ◽  
Martin M. Möckel ◽  
Thomas U. Mayer ◽  
...  
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Motor Domain ◽  
Structural Basis ◽  
General Mechanism ◽  
Antimitotic Drugs ◽  
Crucial Information ◽  
Directed Motility ◽  
Spindle Microtubules

Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end–directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1–bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1–binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.

scholarly journals The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division

2020 ◽  
Author(s):  
Ivan Ramirez ◽  
Ankur A. Gholkar ◽  
Erick F. Velasquez ◽  
Xiao Guo ◽  
Jorge Z. Torres
Keyword(s):  
Cell Division ◽  
Molecular Motors ◽  
Mitotic Spindle ◽  
Regulatory Light Chains ◽  
Cellular Processes ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Early Mitosis

ABSTRACTMyosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane, while myosin MYO2 functions in actomyosin ring contraction to promote cytokinesis. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. Myl5 localized to the mitotic spindle poles and spindle microtubules during early mitosis, an area overlapping with MYO10 localization. Depletion of Myl5 led to defects in chromosome congression and to a slower progression through mitosis. We propose that Myl5 is a novel myosin RLC that is important for cell division.

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