scholarly journals Mitotic Spindle Integrity and Kinetochore Function Linked by the Duo1p/Dam1p Complex

2001 ◽  
Vol 152 (1) ◽  
pp. 197-212 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Maria Enquist-Newman ◽  
Thomas Müller-Reichert ◽  
David G. Drubin ◽  
Georjana Barnes
Keyword(s):  
Mitotic Spindle ◽  
Genetic Interactions ◽  
Yeast Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chromosome Missegregation ◽  
Shared Function ◽  
Mutant Phenotypes ◽  
Bipolar Spindles ◽  
Kinetochore Function

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.

scholarly journals Dad1p, Third Component of the Duo1p/Dam1p Complex Involved in Kinetochore Function and Mitotic Spindle Integrity

2001 ◽  
Vol 12 (9) ◽  
pp. 2601-2613 ◽  
Author(s):  
Maria Enquist-Newman ◽  
Iain M. Cheeseman ◽  
David Van Goor ◽  
David G. Drubin ◽  
Pamela B. Meluh ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
The Novel ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sensitive Allele ◽  
Two Hybrid ◽  
Kinetochore Function

We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

scholarly journals Tension-dependent nucleosome remodeling at the pericentromere in yeast

2012 ◽  
Vol 23 (13) ◽  
pp. 2560-2570 ◽  
Author(s):  
Jolien S. Verdaasdonk ◽  
Ryan Gardner ◽  
Andrew D. Stephens ◽  
Elaine Yeh ◽  
Kerry Bloom
Keyword(s):  
Chromatin Remodeling ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Physical Force ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nucleosome Remodeling ◽  
Nucleosome Dynamics ◽  
Chromosome Arm ◽  
Dna Translocase ◽  
Kinetochore Function

Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.

scholarly journals Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

2001 ◽  
Vol 155 (5) ◽  
pp. 763-774 ◽  
Author(s):  
Jung-seog Kang ◽  
Iain M. Cheeseman ◽  
George Kallstrom ◽  
Soundarapandian Velmurugan ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Related Protein ◽  
Centromeric Dna ◽  
Centromere Protein ◽  
Kinetochore Function

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

scholarly journals Molecular Lesions Associated With Alleles of decapentuplegic Identify Residues Necessary for TGF-β/BMP Cell Signaling in Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 493-505 ◽  
Author(s):  
Kristi Wharton ◽  
Robert P Ray ◽  
Seth D Findley ◽  
Holly E Duncan ◽  
William M Gelbart
Keyword(s):  
Cell Signaling ◽  
Low Temperatures ◽  
Point Mutations ◽  
Molecular Probes ◽  
Genetic Interactions ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mutant Phenotypes

Abstract We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-β homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-β superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embyronic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-β-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.

scholarly journals Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis

2009 ◽  
Vol 284 (24) ◽  
pp. 16501-16512 ◽  
Author(s):  
Kyung Uk Hong ◽  
Hyun-Jun Kim ◽  
Hyo-Sil Kim ◽  
Yeon-Sun Seong ◽  
Kyeong-Man Hong ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Cyclin B1 ◽  
Mutant Form ◽  
Metaphase Chromosomes ◽  
C Terminus ◽  
Mitotic Spindle Assembly ◽  
Bipolar Spindles

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

scholarly journals The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1493-1502
Author(s):  
Richard D Gardner ◽  
Atasi Poddar ◽  
Chris Yellman ◽  
Penny A Tavormina ◽  
M Cristina Monteagudo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Spindle Checkpoint ◽  
Essential Genes ◽  
Gene Fusions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Signaling ◽  
One Step ◽  
Diploid Cells ◽  
Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

scholarly journals Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
Author(s):  
Nallani Vijay Kumar ◽  
Jianbo Yang ◽  
Jitesh K. Pillai ◽  
Swati Rawat ◽  
Carlos Solano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Yeast Protein ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

scholarly journals Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 688-702 ◽  
Author(s):  
J M Ivy ◽  
A J Klar ◽  
J B Hicks
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Blot Analysis ◽  
Transcriptional Control ◽  
Genomic Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Type Genes ◽  
Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

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