scholarly journals Probing the Dynamics and Functions of Aurora B Kinase in Living Cells during Mitosis and Cytokinesis

2002 ◽  
Vol 13 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Maki Murata-Hori ◽  
Masaaki Tatsuka ◽  
Yu-Li Wang
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Aurora B ◽  
Anaphase Onset ◽  
Abnormal Mitosis ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Dividing Cells ◽  
Spindle Midzone ◽  
Green Fluorescent

Aurora B is a protein kinase and a chromosomal passenger protein that undergoes dynamic redistribution during mitosis. We have probed the mechanism that regulates its localization with cells expressing green fluorescent protein (GFP)-tagged wild-type or mutant aurora B. Aurora B was found at centromeres at prophase and persisted until ∼0.5 min after anaphase onset, when it redistributed to the spindle midzone and became concentrated at the equator along midzone microtubules. Depolymerization of microtubules inhibited the dissociation of aurora B from centromeres at early anaphase and caused the dispersion of aurora B from the spindle midzone at late anaphase; however, centromeric association during prometaphase was unaffected. Inhibition of CDK1 deactivation similarly caused aurora B to remain associated with centromeres during anaphase. In contrast, inhibition of the kinase activity of aurora B appeared to have no effect on its interactions with centromeres or initial relocation onto midzone microtubules. Instead, kinase-inactive aurora B caused abnormal mitosis and deactivation of the spindle checkpoint. In addition, midzone microtubule bundles became destabilized and aurora B dispersed from the equator. Our results suggest that microtubules, CDK1, and the kinase activity each play a distinct role in the dynamics and functions of aurora B in dividing cells.

scholarly journals The Abscission Checkpoint: A Guardian of Chromosomal Stability

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3350
Author(s):  
Eleni Petsalaki ◽  
George Zachos
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Nuclear Pore ◽  
Aurora B ◽  
Intercellular Bridge ◽  
Endosomal Sorting ◽  
Daughter Cells ◽  
Chromosomal Passenger ◽  
Dividing Cells ◽  
Chromatin Bridges

The abscission checkpoint contributes to the fidelity of chromosome segregation by delaying completion of cytokinesis (abscission) when there is chromatin lagging in the intercellular bridge between dividing cells. Although additional triggers of an abscission checkpoint-delay have been described, including nuclear pore defects, replication stress or high intercellular bridge tension, this review will focus only on chromatin bridges. In the presence of such abnormal chromosomal tethers in mammalian cells, the abscission checkpoint requires proper localization and optimal kinase activity of the Chromosomal Passenger Complex (CPC)-catalytic subunit Aurora B at the midbody and culminates in the inhibition of Endosomal Sorting Complex Required for Transport-III (ESCRT-III) components at the abscission site to delay the final cut. Furthermore, cells with an active checkpoint stabilize the narrow cytoplasmic canal that connects the two daughter cells until the chromatin bridges are resolved. Unsuccessful resolution of chromatin bridges in checkpoint-deficient cells or in cells with unstable intercellular canals can lead to chromatin bridge breakage or tetraploidization by regression of the cleavage furrow. In turn, these outcomes can lead to accumulation of DNA damage, chromothripsis, generation of hypermutation clusters and chromosomal instability, which are associated with cancer formation or progression. Recently, many important questions regarding the mechanisms of the abscission checkpoint have been investigated, such as how the presence of chromatin bridges is signaled to the CPC, how Aurora B localization and kinase activity is regulated in late midbodies, the signaling pathways by which Aurora B implements the abscission delay, and how the actin cytoskeleton is remodeled to stabilize intercellular canals with DNA bridges. Here, we review recent progress toward understanding the mechanisms of the abscission checkpoint and its role in guarding genome integrity at the chromosome level, and consider its potential implications for cancer therapy.

scholarly journals INCENP–aurora B interactions modulate kinase activity and chromosome passenger complex localization

2009 ◽  
Vol 187 (5) ◽  
pp. 637-653 ◽  
Author(s):  
Zhenjie Xu ◽  
Hiromi Ogawa ◽  
Paola Vagnarelli ◽  
Jan H. Bergmann ◽  
Damien F. Hudson ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Aurora B ◽  
Activity Levels ◽  
Checkpoint Response ◽  
C Terminus ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Low Levels ◽  
Chromosome Passenger Complex

Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

scholarly journals Disassembly of actin and keratin networks by Aurora B kinase at the midplane of cleaving Xenopus laevis eggs

2019 ◽  
Author(s):  
Christine M. Field ◽  
James F. Pelletier ◽  
Timothy J. Mitchison
Keyword(s):  
Xenopus Laevis ◽  
Kinase Activity ◽  
Flow Patterns ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Chromosomal Passenger Complex ◽  
Chromosomal Passenger ◽  
Dividing Cells ◽  
Local Softening

AbstractWe investigated how bulk cytoplasm prepares for cytokinesis in Xenopus laevis eggs, which are large, rapidly dividing cells. The egg midplane is demarcated by Chromosomal Passenger Complex (CPC) localized on microtubule bundles between asters. Using an extract system and intact eggs we found that local kinase activity of the AURKB subunit of the CPC caused disassembly of F-actin and keratin between asters, and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ~40 μm without microtubules, and much farther with microtubules present, due to CPC auto-activation. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. This may help sister centrosomes move apart after mitosis, prepare a soft path for furrow ingression and/or release G-actin to build the furrow cortex.

scholarly journals Phosphatase Inhibitor-2 Balances Protein Phosphatase 1 and Aurora B Kinase for Chromosome Segregation and Cytokinesis in Human Retinal Epithelial Cells

2008 ◽  
Vol 19 (11) ◽  
pp. 4852-4862 ◽  
Author(s):  
Weiping Wang ◽  
P. Todd Stukenberg ◽  
David L. Brautigan
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Multinucleated Cells ◽  
Multipolar Spindles ◽  
Spindle Midzone ◽  
Green Fluorescent

Mitosis in Saccharomyces cerevisiae depends on IPL1 kinase, which genetically interacts with GLC8. The metazoan homologue of GLC8 is inhibitor-2 (I-2), but its function is not understood. We found endogenous and ectopic I-2 localized to the spindle, midzone, and midbody of mitotic human epithelial ARPE-19 cells. Knockdown of I-2 by RNA interference produced multinucleated cells, with supernumerary centrosomes, multipolar spindles and lagging chromosomes during anaphase. These defects did not involve changes in levels of protein phosphatase-1 (PP1), and the multinuclear phenotype was rescued by overexpression of I-2. Appearance of multiple nuclei and supernumerary centrosomes required progression through the cell cycle and I-2 knockdown cells failed cytokinesis, as observed by time-lapse microscopy. Inhibition of Aurora B by hesperadin produced multinucleated cells and reduced H3S10 phosphorylation. I-2 knockdown enhanced this latter effect. Partial knockdown of PP1Cα prevented multiple nuclei caused by either knockdown of I-2 or treatment with hesperadin. Expression of enhanced green fluorescent protein-I-2 or hemagglutinin-I-2 made cells resistant to hesperadin. We propose that I-2 acts to enhance Aurora B by inhibiting specific PP1 holoenzymes that dephosphorylate Aurora B substrates necessary for chromosome segregation and cytokinesis. Conserved together throughout eukaryotic evolution, I-2, PP1 and Aurora B function interdependently during mitosis.

scholarly journals One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Joseph R. Owen ◽  
Sadie L. Hennig ◽  
Bret R. McNabb ◽  
Tamer A. Mansour ◽  
Justin M. Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bos Taurus ◽  
Single Copy ◽  
Blastocyst Stage ◽  
Target Site ◽  
End Joining ◽  
Bull Calf ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Donor Template

Abstract Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

Insulin-stimulated trafficking of ENaC in renal cells requires PI 3-kinase activity

2003 ◽  
Vol 284 (6) ◽  
pp. C1645-C1653 ◽  
Author(s):  
Bonnie L. Blazer-Yost ◽  
Michail A. Esterman ◽  
Chris J. Vlahos
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Apical Cell ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Insulin Stimulation ◽  
Α Subunit ◽  
Clonal Lines ◽  
Green Fluorescent ◽  
Apical Membranes

αENaC-EGFP (enhanced green fluorescent protein-tagged α-subunit of the epithelial Na+ channel) stably transfected clonal lines derived from the A6 parental cell line were used to study the physical mechanisms of insulin-stimulated Na+ transport. Within 1 min of insulin stimulation, ENaC migrates from a diffuse cytoplasmic localization to the apical and lateral membranes. Concurrently, after insulin stimulation, phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC on the lateral but not apical membrane. An inhibitor of PI 3-kinase, LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but does alter the intracellular site of the colocalization, preventing the translocation of ENaC to the lateral and apical membranes. These data show that insulin stimulation causes the migration of ENaC to the lateral and apical cell membranes and that this trafficking is dependent on PI 3-kinase activity.

scholarly journals Changes in the Min Oscillation Pattern before and after Cell Birth

2010 ◽  
Vol 192 (16) ◽  
pp. 4134-4142 ◽  
Author(s):  
Jennifer R. Juarez ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
The Other ◽  
Cell Pole ◽  
Daughter Cells ◽  
Division Site ◽  
Before And After ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

scholarly journals PLK1 plays dual roles in centralspindlin regulation during cytokinesis

2019 ◽  
Vol 218 (4) ◽  
pp. 1250-1264 ◽  
Author(s):  
Ingrid E. Adriaans ◽  
Angika Basant ◽  
Bas Ponsioen ◽  
Michael Glotzer ◽  
Susanne M.A. Lens
Keyword(s):  
Small Gtpase ◽  
The Other ◽  
Aurora B ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Dual Roles ◽  
Early Step ◽  
Rhoa Activation ◽  
Anaphase Onset ◽  
Spindle Midzone

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.

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