Ybp2 Associates with the Central Kinetochore of Saccharomyces cerevisiae and Mediates Proper Mitotic Progression

The spindle checkpoint ensures the accurate segregation of chromosomes by monitoring the status of kinetochore attachment to microtubules. Simultaneous mutations in one of several kinetochore and cohesion genes and a spindle checkpoint gene cause a synthetic-lethal or synthetic-sick phenotype. A synthetic genetic array (SGA) analysis using a mad2Δ query mutant strain of yeast identified YBP2, a gene whose product shares sequence similarity with the product of YBP1, which is required for H2O2-induced oxidation of the transcription factor Yap1. ybp2Δ was sensitive to benomyl and accumulated at the mitotic stage of the cell cycle. Ybp2 physically associates with proteins of the COMA complex (Ctf19, Okp1, Mcm21, and Ame1) and 3 components of the Ndc80 complex (Ndc80, Nuf2, and Spc25 but not Spc24) in the central kinetochore and with Cse4 (the centromeric histone and CENP-A homolog). Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA. Furthermore, ybp2Δ showed synthetic-sick interactions with mutants of the genes that encode the COMA complex components. Ybp2 seems to be part of a macromolecular kinetochore complex and appears to contribute to the proper associations among the central kinetochore subcomplexes and the kinetochore-specific nucleosome

FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion

BACKGROUND: Phycobilisomes (PBsomes) are the extrinsic antenna complexes upon the photosynthetic membranes in red algae and most cyanobacteria. The PBsomes in the cyanobacteria has been proposed to present high lateral mobility on the thylakoid membrane surface. In contrast, direct measurement of PBsome motility in red algae has been lacking so far. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we investigated the dynamics of PBsomes in the unicellular red alga Porphyridium cruentum in vivo and in vitro, using fluorescence recovery after photobleaching (FRAP). We found that part of the fluorescence recovery could be detected in both partially- and wholly-bleached wild-type and mutant F11 (UTEX 637) cells. Such partial fluorescence recovery was also observed in glutaraldehyde-treated and betaine-treated cells in which PBsome diffusion should be restricted by cross-linking effect, as well as in isolated PBsomes immobilized on the glass slide. CONCLUSIONS/SIGNIFICANCE: On the basis of our previous structural results showing the PBsome crowding on the native photosynthetic membrane as well as the present FRAP data, we concluded that the fluorescence recovery observed during FRAP experiment in red algae is mainly ascribed to the intrinsic photoprocesses of the bleached PBsomes in situ, rather than the rapid diffusion of PBsomes on thylakoid membranes in vivo. Furthermore, direct observations of the fluorescence dynamics of phycoerythrins using FRAP demonstrated the energetic decoupling of phycoerythrins in PBsomes against strong excitation light in vivo, which is proposed as a photoprotective mechanism in red algae attributed by the PBsomes in response to excess light energy

Bub1-Mediated Adaptation of the Spindle Checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore–microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest

Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex

A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells

Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation

Stressful situation if CENP-A not front and CENter

The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation

Effects of perceived cocaine availability on subjective and objective responses to the drug

<p>Abstract</p> <p>Rationale</p> <p>Several lines of evidence suggest that cocaine expectancy and craving are two related phenomena. The present study assessed this potential link by contrasting reactions to varying degrees of the drug's perceived availability.</p> <p>Method</p> <p>Non-treatment seeking individuals with cocaine dependence were administered an intravenous bolus of cocaine (0.2 mg/kg) under 100% ('unblinded'; N = 33) and 33% ('blinded'; N = 12) probability conditions for the delivery of drug. Subjective ratings of craving, high, rush and low along with heart rate and blood pressure measurements were collected at baseline and every minute for 20 minutes following the infusions.</p> <p>Results</p> <p>Compared to the 'blinded' subjects, their 'unblinded' counterparts had similar craving scores on a multidimensional assessment several hours before the infusion, but reported higher craving levels on a more proximal evaluation, immediately prior to the receipt of cocaine. Furthermore, the 'unblinded' subjects displayed a more rapid onset of high and rush cocaine responses along with significantly higher cocaine-induced heart rate elevations.</p> <p>Conclusion</p> <p>These results support the hypothesis that cocaine expectancy modulates subjective and objective responses to the drug. Provided the important public health policy implications of heavy cocaine use, health policy makers and clinicians alike may favor cocaine craving assessments performed in the settings with access to the drug rather than in more neutral environments as a more meaningful marker of disease staging and assignment to the proper level of care.</p

Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades