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 PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

2014 ◽  
Vol 206 (7) ◽  
pp. 833-842 ◽  
Author(s):  
Antonio Espert ◽  
Pelin Uluocak ◽  
Ricardo Nunes Bastos ◽  
Davinderpreet Mangat ◽  
Philipp Graab ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Eukaryotic Cells ◽  
Aurora B ◽  
Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

scholarly journals CSC-1

2003 ◽  
Vol 161 (2) ◽  
pp. 229-236 ◽  
Author(s):  
Alper Romano ◽  
Annika Guse ◽  
Ivica Krascenicova ◽  
Heinke Schnabel ◽  
Ralf Schnabel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Analysis ◽  
Chromosome Segregation ◽  
Kinase Activity ◽  
Aurora B ◽  
Aurora B Kinase ◽  
C Elegans ◽  
The Individual

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

scholarly journals Differentiation of Cytoplasmic and Meiotic Spindle Assembly MCAK Functions by Aurora B-dependent Phosphorylation

2004 ◽  
Vol 15 (6) ◽  
pp. 2895-2906 ◽  
Author(s):  
Ryoma Ohi ◽  
Tanuj Sapra ◽  
Jonathan Howard ◽  
Timothy J. Mitchison
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Meiotic Spindle ◽  
Aurora B ◽  
Dependent Manner ◽  
Dynamic Nature ◽  
Spindle Microtubule ◽  
Bipolar Spindles

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.

scholarly journals Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

2018 ◽  
Vol 218 (2) ◽  
pp. 455-473 ◽  
Author(s):  
Cai Tong Ng ◽  
Li Deng ◽  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Depolymerization ◽  
Yeast Chromosome ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Electron Cryotomography ◽  
Binding Position

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

scholarly journals Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

2018 ◽  
Author(s):  
Anna K. de Regt ◽  
Charles L. Asbury ◽  
Sue Biggins
Keyword(s):  
Chromosome Segregation ◽  
Direct Evidence ◽  
Underlying Mechanism ◽  
Aurora B ◽  
Trial And Error ◽  
Spindle Poles ◽  
Pulling Forces ◽  
Low Tension

Introduction / AbstractChromosome segregation requires large macromolecular structures called kinetochores to attach dynamic microtubules from opposite spindle poles1,2. Attachments are made iteratively, through a trial-and-error process, and proper attachments come under tension from the pulling forces of microtubules3,4. However, if sister kinetochores bind microtubules from the same pole1,2, these defective attachments lack tension and must be destabilized to give another chance for proper attachments to form. This vital error correction process requires Aurora B kinase, which phosphorylates kinetochores lacking tension to reduce their affinity for microtubules5-11. An unresolved question is how Aurora B distinguishes the level of tension on kinetochores. There are conflicting reports on the underlying mechanism12-16, owing in part to the difficulties of manipulating kinetochore tension in vivo and distinguishing kinase from opposing phosphatase activity. To address these issues, we have reconstituted Aurora B-triggered kinetochore detachment in an in vitro optical trapping-based flow assay. Here, we test an outstanding model by determining whether kinetochore tension is sufficient to prevent kinase-triggered detachments. Strikingly, Aurora B detaches kinetochores from microtubules under both high and low tension, providing direct evidence that the kinase does not distinguish correct versus incorrect attachments by recognizing tension-dependent changes in the conformation of its kinetochore substrates.

scholarly journals Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

2008 ◽  
Vol 19 (10) ◽  
pp. 4534-4544 ◽  
Author(s):  
Jérémie Gaillard ◽  
Emmanuelle Neumann ◽  
Daniel Van Damme ◽  
Virginie Stoppin-Mellet ◽  
Christine Ebel ◽  
...  
Keyword(s):  
Preprophase Band ◽  
Spindle Microtubule ◽  
Microtubule Associated Proteins ◽  
Late Anaphase ◽  
Associated Proteins ◽  
Planar Network ◽  
Spindle Midzone ◽  
Spindle Microtubules

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.

scholarly journals A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

2018 ◽  
Author(s):  
Matthew P. Miller ◽  
Rena K. Evans ◽  
Alex Zelter ◽  
Elisabeth A. Geyer ◽  
Michael J. MacCoss ◽  
...  
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Critical Aspect ◽  
Daughter Cells ◽  
Low Tension ◽  
Kinetochore Function ◽  
Segregation Of Chromosomes

ABSTRACTAccurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be destabilized. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. This pathway’s contribution to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes error correction defects in vivo. We also show that this intrinsic tension-sensing pathway functions in concert with the Aurora B-mediated pathway. Together, our work indicates that cells employ at least two pathways to ensure biorientation and the accuracy of chromosome segregation.

scholarly journals Aurora B Overexpression Causes Aneuploidy and p21Cip1Repression during Tumor Development

2015 ◽  
Vol 35 (20) ◽  
pp. 3566-3578 ◽  
Author(s):  
Alejandra González-Loyola ◽  
Gonzalo Fernández-Miranda ◽  
Marianna Trakala ◽  
David Partida ◽  
Kumiko Samejima ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Cultured Cells ◽  
Tumor Development ◽  
Inverse Correlation ◽  
Aurora Kinase ◽  
Tumor Formation ◽  
Aurora B ◽  
Aurora Kinase B

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogenein vivois not established. Here, we report a new mouse model in which expression of the endogenousAurkblocus can be inducedin vitroandin vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora Bin vivoresults in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21Cip1in vitroandin vivo, in line with an inverse correlation between Aurora B and p21Cip1expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21Cip1.

scholarly journals FORMULATION AND IN VITRO, IN VIVO EVALUATION OF PRONIOSOMAL GEL OF NEOMYCIN SULPHATE

2019 ◽  
pp. 156-163
Author(s):  
AMOL SHETE ◽  
PRIYANKA THORAT ◽  
RAJENDRA DOIJAD ◽  
SACHIN SAJANE
Keyword(s):  
Phase Separation ◽  
Skin Irritation ◽  
Entrapment Efficiency ◽  
Separation Method ◽  
Gelling Agent ◽  
In Vivo Evaluation ◽  
Skin Delivery ◽  
Span 60

Objective: The objectives of present investigation were to prepare and evaluate proniosomes of neomycin sulphate (NS) by coacervation phase separation method by using sorbitan monostearate (span 60) and lecithin as a surfactant to increase the penetration through the skin and study the effect of concentration of the same. Methods: Proniosomes of neomycin sulphate (NS) were prepared by coacervation phase separation method by using span 60 and lecithin. The effect of concentration of span 60 and lecithin was studied by factorial design. The prepared proniosomes were converted to gel by using carbopol as a gelling agent. The prepared formulations were evaluated for entrapment efficiency, in vitro drug diffusion, in vitro antibacterial activity and in vivo skin irritation test etc. Results: All Formulation showed the percentage entrapment efficiency in the range 38.31±0.05% to 77.96±0.06%, good homogeneity and gel was easily spreadable with minimal of shear. Optimized formulation showed enhanced rate of diffusion in vitro, increase in zone of inhibition against staphylococcus aureus, no skin irritation and showed good stability. Conclusion: The results of present study indicates that proniosomal gel formulated by using combination of span 60, Lecithin, cholesterol can be used to enhance skin delivery of NS because of excellent permeation of drug. Developed proniosomal gel formulation was promising carrier for NS

scholarly journals Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 915
Author(s):  
Jazelli Mueterthies ◽  
Davit A. Potoyan
Keyword(s):  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Bodies ◽  
Disordered Proteins ◽  
Solvent Exposure ◽  
Ion Release ◽  
Dimer Formation ◽  
Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

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