scholarly journals Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

2010 ◽  
Vol 188 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Franco J. Vizeacoumar ◽  
Nydia van Dyk ◽  
Frederick S.Vizeacoumar ◽  
Vincent Cheung ◽  
Jingjing Li ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Regulatory Networks ◽  
Genetic Interaction ◽  
High Content Screening ◽  
Cellular Functions ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Chromosomal Passenger Proteins ◽  
Spindle Disassembly ◽  
Passenger Proteins

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation

1991 ◽  
Vol 98 (4) ◽  
pp. 443-461 ◽  
Author(s):  
W.C. Earnshaw ◽  
C.A. Cooke
Keyword(s):  
Structural Changes ◽  
Metaphase Plate ◽  
Maximum Diameter ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Intracellular Location ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Chromosomal Passenger Proteins ◽  
Passenger Proteins

The INCENPs are two polypeptides of 135 × 10(3) and 150 × 10(3) Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In ‘early’ and ‘early/mid’ metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During ‘mid/late’ metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 microns in diameter and approximately 4 microns beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of ‘chromosomal passenger’ proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.

Megakaryocytes express functional Aurora-B kinase in endomitosis

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1017-1024 ◽  
Author(s):  
Amy E. Geddis ◽  
Kenneth Kaushansky
Keyword(s):  
Aurora Kinase ◽  
Aurora B ◽  
Aurora A Kinase ◽  
Aurora B Kinase ◽  
Late Anaphase ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Chromosomal Passenger Proteins ◽  
Diploid Cells ◽  
Passenger Proteins

AbstractEndomitosis (EnM) in megakaryocytes (MKs) is characterized by abortion of mitosis in late anaphase and failure of cytokinesis; subsequent reinitiation of DNA synthesis results in polyploidy. Ablation of chromosomal passenger proteins including Aurora-B kinase causes defects in late anaphase and cytokinesis in diploid cells; thus one hypothesis is that the expression or function of these proteins in polyploid MKs is abnormal. It has been reported that Aurora-B kinase mRNA is decreased in polyploid megakaryocytic cells, suggesting that deficiency of Aurora-B kinase is responsible for EnM. We examined the localization of Aurora-B kinase and additional members of the chromosomal passenger protein and aurora kinase families in MKs. We found that in EnM MKs (1) Aurora-B kinase is present and appropriately localized to centromeres in early EnM; (2) in low-ploidy human MKs, centromeric localization of survivin and inner centromere protein (INCENP) can also be demonstrated; (3) the function of Aurora-B kinase, as measured by Ser10 phosphorylation of histone H3, is intact; and (4) aurora-A kinase localizes appropriately to centrosomes in EnM. These results suggest that EnM MKs appropriately express functional Aurora-B kinase and related proteins in early anaphase, making a simple deficiency of this protein an unlikely explanation for polyploidy in this cell type.

scholarly journals The Mitotic Exit Network Mob1p-Dbf2p Kinase Complex Localizes to the Nucleus and Regulates Passenger Protein Localization

2005 ◽  
Vol 16 (12) ◽  
pp. 5465-5479 ◽  
Author(s):  
Jan Stoepel ◽  
Michelle A. Ottey ◽  
Cornelia Kurischko ◽  
Philip Hieter ◽  
Francis C. Luca
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Protein Localization ◽  
Mitotic Exit ◽  
Mitotic Exit Network ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Chromosomal Passenger Proteins ◽  
Kinetochore Region ◽  
Passenger Proteins ◽  
Passenger Protein

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates CDK inactivation, cytokinesis and G1 gene transcription. The MEN Cdc14p phosphatase is sequestered in the nucleolus and transiently released in early anaphase and telophase. Cdc14p mediates mitotic exit by dephosphorylating Cdk1p substrates and promoting Cdk1p inactivation. Cdc14p also regulates the localization of chromosomal passenger proteins, which redistribute from kinetochores to the mitotic spindle during anaphase. Here we present evidence that the MEN protein kinase complex Mob1p-Dbf2p localizes to mitotic nuclei and partially colocalizes with Cdc14p and kinetochore proteins. Chromatin immunoprecipitation (ChIP) experiments reveal that Mob1p, Dbf2p, and Cdc14p associate with centromere DNA and require the centromere binding protein Ndc10p for this association. We establish that Mob1p is essential for maintaining the localization of Aurora, INCENP, and Survivin chromosomal passenger proteins on anaphase spindles, whereas Cdc14p and the Mob1p-Dbf2p-activating kinase Cdc15p are required for establishing passenger protein localization on the spindle. Moreover, Mob1p, but not Cdc15p, is required for dissociating Aurora from the kinetochore region. These findings reveal kinetochores as sites for MEN signaling and implicate MEN in coordinating chromosome segregation and/or spindle integrity with mitotic exit and cytokinesis via regulation of chromosome passenger proteins.

scholarly journals Shugoshin 2 Regulates Localization of the Chromosomal Passenger Proteins in Fission Yeast Mitosis

2007 ◽  
Vol 18 (5) ◽  
pp. 1657-1669 ◽  
Author(s):  
Vincent Vanoosthuyse ◽  
Sergey Prykhozhij ◽  
Kevin G. Hardwick
Keyword(s):  
Fission Yeast ◽  
Spindle Checkpoint ◽  
Aurora B ◽  
Checkpoint Activation ◽  
C Terminus ◽  
Chromosomal Passenger ◽  
Chromosomal Passenger Proteins ◽  
Passenger Proteins ◽  
Meiosis I ◽  
Mitotic Cells

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells.

scholarly journals Bir1 Is Required for the Tension Checkpoint

2009 ◽  
Vol 20 (3) ◽  
pp. 915-923 ◽  
Author(s):  
Michelle M. Shimogawa ◽  
Per O. Widlund ◽  
Michael Riffle ◽  
Michael Ess ◽  
Trisha N. Davis
Keyword(s):  
Aurora B ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Checkpoint Response ◽  
Temperature Sensitive Mutant ◽  
The Third ◽  
Chromosomal Passenger ◽  
Chromosomal Passenger Proteins ◽  
Passenger Proteins

The Saccharomyces cerevisiae chromosomal passenger proteins Ipl1 (Aurora B) and Sli15 (INCENP) are required for the tension checkpoint, but the role of the third passenger, Bir1, is controversial. We have isolated a temperature-sensitive mutant (bir1-107) in the essential C-terminal region of Bir1 known to be required for binding to Sli15. This allele reveals a checkpoint function for Bir1. The mutant displays a biorientation defect, a defective checkpoint response to lack of tension, and an inability to detach mutant kinetochores. Ipl1 localizes to aberrant foci when Bir1 localization is disrupted in the bir1-107 mutant. Thus, one checkpoint role of Bir1 is to properly localize Ipl1 and allow detachment of kinetochores. Quantitative analysis indicates that the chromosomal passengers colocalize with kinetochores in G1 but localize between kinetochores that are under tension. Bir1 localization to kinetochores is maintained in an mcd1-1 mutant in the absence of tension. Our results suggest that the establishment of tension removes Ipl1, Bir1, and Sli15, and their kinetochore detachment activity, from the vicinity of kinetochores and allows cells to proceed through the tension checkpoint.

The role of centromere-binding factor 3 (CBF3) in spindle stability, cytokinesis, and kinetochore attachment

2005 ◽  
Vol 83 (6) ◽  
pp. 696-702 ◽  
Author(s):  
David Bouck ◽  
Kerry Bloom
Keyword(s):  
Essential Role ◽  
Anaphase Onset ◽  
Binding Factor ◽  
Chromosomal Passenger ◽  
Chromosomal Passenger Proteins ◽  
Spindle Midzone ◽  
Spindle Stability ◽  
Passenger Proteins ◽  
Passenger Protein

The spindle midzone is critical for spindle stability and cytokinesis. Chromosomal passenger proteins relocalize from chromosomes to the spindle midzone after anaphase onset. The recent localization of the inner-kinetochore, centromere-binding factor 3 (CBF3) complex to the spindle midzone in budding yeast has led to the discovery of novel functions for this complex in addition to its essential role at kinetochores. In G1/S cells, CBF3 components are detected along dynamic microtubules, where they can "search-and-capture" newly replicated centromeres. During anaphase, CBF3 is transported to the microtubule plus-ends of the spindle midzone. Consistent with this localization, cells containing a mutation in the CBF3 subunit Ndc10p show defects in spindle stability during anaphase. In addition, ndc10-1 cells show defects during cytokinesis, resulting in a defect in cell abscission. These results highlight the importance of midzone-targeted proteins in coordinating mitosis with cell division. Here we discuss these findings and explore the significance of CBF3 transport to microtubule plus-ends at the spindle midzone.Key words: spindle midzone, passenger protein, inner centromere protein (INCENP), microtubule plus-end.

scholarly journals Basal-Type Breast Cancer Stem Cells Over-Express Chromosomal Passenger Complex Proteins

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 709
Author(s):  
Angela Schwarz-Cruz y Celis ◽  
Gisela Ceballos-Cancino ◽  
Karla Vazquez-Santillan ◽  
Magali Espinosa ◽  
Cecilia Zampedri ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cancer Progression ◽  
Cellular Growth ◽  
Analysis Tool ◽  
Chromosomal Passenger ◽  
Chromosomal Passenger Proteins ◽  
Passenger Proteins

(1) Aim: In the present paper we analyzed the transcriptome of CSCs (Cancer Stem Cells), in order to find defining molecular processes of breast cancer. (2) Methods: We performed RNA-Seq from CSCs isolated from the basal cell line MDA-MB-468. Enriched processes and networks were studied using the IPA (Ingenuity Pathway Analysis) tool. Validation was performed with qRT-PCR and the analysis of relevant genes was evaluated by overexpression, flow cytometry and in vivo zebrafish studies. Finally, the clinical relevance of these results was assessed using reported cohorts. (3) Results: We found that CSCs presented marked differences from the non-CSCs, including enrichment in transduction cascades related to stemness, cellular growth, proliferation and apoptosis. Interestingly, CSCs overexpressed a module of co-regulated Chromosomal Passenger Proteins including BIRC5 (survivin), INCENP and AURKB. Overexpression of BIRC5 increased the number of CSCs, as assessed by in vitro and in vivo zebrafish xenotransplant analyses. Analysis of previously published cohorts showed that this co-regulated module was not only overexpressed in basal breast tumors but also associated with relapse-free and overall survival in these patients. (4) Conclusions: These results underline the importance of Cancer Stem Cells in breast cancer progression and point toward the possible use of chromosomal passenger proteins as prognostic factors.

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