Precocious activation of APC/C-Cdh1 at pre-anaphase causes genome instability

Faithful chromosome segregation and thereby accurate gene transmission are crucial for all organisms. Until proper attachment of the mitotic spindle to the kinetochore is established, the ubiquitin ligase (E3) Cdc20-activated APC/C (anaphase promoting complex/cyclosome) is repressed by the spindle assembly checkpoint (SAC) and sister chromatin cohesion is protected. Mutants defective in SAC fail to arrest at metaphase even in the presence of damaged microtubules. Interestingly, a similar phenomenon occurs in yeast cells defective in Bub2, a negative factor of the mitotic exit network (MEN), which is required for telophase onset, although its precise molecular mechanism is unknown. Here, we show that chromosome missegregation occurs frequently in bub2∆ cells in the presence of damaged microtubules. The loss of Bub2 caused precocious activation of APC/C-Cdh1/Hct1 at pre-anaphase, leading to securin degradation and then separase-mediated cohesin cleavage. Overexpression of CDH1 and CDC14, encoding Cdc14 phosphatase, at pre-anaphase similarly caused chromosome missegregation. Thus, sequential activation of APC/C-Cdc20 and then APC/C-Cdh1 is critical for precise chromosome segregation and precocious activation of APC/C-Cdh1 at pre-anaphase causes genomic instability. Since degradation of human securin is also mediated by APC/C-Cdc20 and APC/C-Cdh1, this study predicts that precocious activation APC/C-Cdh1 in human cells similarly causes genomic instability, and thereby cell death or tumorigenesis

Immunolocalization of antioxidant enzymes in high-pressure frozen root and stem nodules of Sesbania rostrata

34 Pags.- 7 Figs.- 1 Tabl. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-8137The activities and localizations of superoxide dismutases (SODs) were compared in root and stem nodules of the semi-aquatic legume Sesbania rostrata using gel-activity assays and immunogold labelling, respectively. Nodules were fixed by high-pressure freezing and dehydrated by freeze substitution. Stem nodules showed more total and specific SOD activities than root nodules because of the presence of chloroplastic CuZnSOD. Most of the total SOD activity of stem and root nodules resulted from 'cytosolic' CuZnSOD, localized in the cytoplasm and chromatin, and from MnSOD in the bacteroids and in the mitochondria of vascular tissue. FeSOD was present in nodule plastids and in leaf chloroplasts, and was found to be associated with chromatin. Superoxide production was detected histochemically in the vascular bundles and in the infected tissue of stem and root nodules, whereas peroxide accumulation was observed in the cortical cell walls and intercellular spaces, as well as within the infection threads of both nodule types. These data suggest a role of CuZnSOD and FeSOD in protecting nuclear DNA from reactive oxygen species and/or in modulating gene activity. The enhanced levels of CuZnSOD, MnSOD and superoxide production in vascular bundle cells are consistent with a role of CuZnSOD and superoxide in the lignification of xylem vessels, but also suggest additional functions in coping with superoxide production by the high respiratory activity of parenchyma cells.This work was supported by the Royal Society (UK), Ministerio de Educación y Ciencia-Fondos Europeos de Desarrollo Regional (AGL2005-01404 and AGL2008-01298) and Gobierno de Aragón (group A53). E.K.J. thanks the Royal Society (UK) and Gobierno de Aragón-Caja Inmaculada (Spain) for funding a sabbatical leave (‘Programa Europa’). M.C.R. was the recipient of a postdoctoral contract (Program I3P) of Consejo Superior de Investigaciones Científicas-Fondo Social Europeo.Peer Reviewe

APC/C-Cdh1-dependent anaphase and telophase progression during mitotic slippage

<p>Abstract</p> <p>Background</p> <p>The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.</p> <p>Results</p> <p>Here we describe mitotic slippage in yeast <it>bub2Δ </it>mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.</p> <p>Conclusions</p> <p>The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.</p

TORC1 regulates autophagy induction in response to proteotoxic stress in yeast and human cells

Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells

Increase in Cytochorome c and a 11.9 kDa protein in Submerged Rice Seedlings after Exposure to Air

To examine the changes in cytocrome c content in submerged rice seedlings after exposure to air, antiserum was prepared against purified cytocrome c from rice bran. Western blottong analysis revealed that cytochrome c was detected 6 h after exposure to air, but not detected in submerged rice seedling. On a fresh weight basis, the same level of cytochrome c as that of the aerobic control was found in the 24-h-air adapted seedlings. judging from the high A408/A280 ratio (4.66),the cytochrome c preparation used as antigen was considered to be well purified. However, the antiserum reacted other several polypeptides. One of them reacted more strongly against the antisermu than cytochrome c and its molecular weight was estimated as 11.9 kDa. The polypeptide increased during air adaptation and the levels found in both submerged seedlings and aerobic control were lower than that in 24-h-air-adapted seedlings.イネ水中芽生えを空気に触れさせた後のチトクロムc含量の変動を調べるために、米ぬかから精製したチトクロムcを用いて抗チトクロムc血清を作成した。ウェスタンプロット分析によると、水中芽生えにはチトクロムcは検出されなかったが、空気に触れてから6時間以降に検出された。酸素適応24時間の芽生えには生重量あたりで好気対照と同レベルのチトクロムcが存在した。抗血清作成の抗原として用いたチトクロムc標品は、A408/A280比の値（4.66）が高いことから、よく精製されていると考えられるが、抗血清はいくつかの他のポリペプチドと反応した。そのうちの一つはチトクロムcよりも抗血清に強く反応し、その分子量は11.9kDaであった。このポリペプチドは酸素適応過程で増加し、水中芽生えや好気対照では24時間酸素適応芽生えよりも少なかった

CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy

Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear-vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA-nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins

Apoptosis at Inflection Point in Liquid Culture of Budding Yeasts

Budding yeasts are highly suitable for aging studies, because the number of bud scars (stage) proportionally correlates with age. Its maximum stages are known to reach at 20–30 stages on an isolated agar medium. However, their stage dynamics in a liquid culture is virtually unknown. We investigate the population dynamics by counting scars in each cell. Here one cell division produces one new cell and one bud scar. This simple rule leads to a conservation law: “The total number of bud scars is equal to the total number of cells.” We find a large discrepancy: extremely fewer cells with over 5 scars than expected. Almost all cells with 6 or more scars disappear within a short period of time in the late log phase (corresponds to the inflection point). This discrepancy is confirmed directly by the microscopic observations of broken cells. This finding implies apoptosis in older cells (6 scars or more)

A Quantitative Analysis of Geographic Color Variation in Two Geotrupes Dung Beetles

We conducted a quantitative analysis of geographic color variation in two species of dung beetles: Geotrupes auratus and G. laevistriatus. The reflectance of the dorsal surfaces was measured from 300 to 700 nm using a spectrophotometer. The reflectance curves for both beetles were bimodal; there were two distinct peaks, namely, the α peak, between 400 and 700 nm, and the β peak at around 300 nm. A stepwise discriminant analysis indicated that geographic color variation in Geotrupes beetles was primarily characterized by a shift of the α peak. Using beetles from three locations, we compared the wavelength (nm) of the α peak (λmax(α)) and its reflectance intensity (R(α)) to investigate sex and population differences. Intraspecific geographic variation in coloration was effectively detected by discriminant analysis of spectral reflectance curves. Our results showed that G. auratus and G. laevistriatus had similar coloration within each sampling location. Our study also revealed hidden sex differences in R(α); R(α) of males were significantly higher than those of females in both species. Since the dorsal surface of the beetles shows remarkable color variation, and coloration can be assessed objectively using reflectance spectra, Geotrupes beetles may be good model organisms to investigate geographic color variation

Evolutionary conservation of TORC1 components, TOR, Raptor, and LST8, between rice and yeast

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