Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast

Background

The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/C^Ama1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.

Findings

Here we show that Ama1p is a substrate of APC/C^Cdc20 but not APC/C^Cdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/C^Cdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/C^Ama1-mediated Cdc20p degradation.

Conclusions

Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.

     

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C^Cdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C^Cdc20 substrate destruction.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis

Background

Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCF^β-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.

Results

We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.

Conclusions

These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

     

Maximizing power generation from dark fermentation effluents in microbial fuel cell by selective enrichment of exoelectrogens and optimization of anodic operational parameters

Objective

To selectively enrich an electrogenic mixed consortium capable of utilizing dark fermentative effluents as substrates in microbial fuel cells and to further enhance the power outputs by optimization of influential anodic operational parameters.

Results

A maximum power density of 1.4 W/m³ was obtained by an enriched mixed electrogenic consortium in microbial fuel cells using acetate as substrate. This was further increased to 5.43 W/m³ by optimization of influential anodic parameters. By utilizing dark fermentative effluents as substrates, the maximum power densities ranged from 5.2 to 6.2 W/m³ with an average COD removal efficiency of 75% and a columbic efficiency of 10.6%.

Conclusion

A simple strategy is provided for selective enrichment of electrogenic bacteria that can be used in microbial fuel cells for generating power from various dark fermentative effluents.

     

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Introduction

Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.

Objectives

To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.

Methods

We present a double-labelling LC–MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.

Results

While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.

Conclusions

Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.

Graphical abstract

Open image in new window

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.     

Multiple Anaphase-promoting Complex/Cyclosome Degrons Mediate the Degradation of Human Sgo1

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G₁/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C^Cdh1) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs in two steps in vertebrate cells (1-3). In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and removed from chromosome arms (1, 4). Then, cleavage of centromeric cohesin by separase takes place at the metaphase-to-anaphase transition to allow sister-chromatid separation (5). The shugoshin (Sgo) family of proteins plays an important role in the protection of centromeric cohesion (6, 7). Human cells depleted of Sgo1 by RNAi undergo massive chromosome missegregation (8-11). In cells with compromised Sgo1 function, centromeric cohesin is improperly phosphorylated and removed (4, 11), resulting in premature sister-chromatid separation. It has been shown recently that Sgo1 collaborates with PP2A to counteract the action of Plk1 and other mitotic kinases and to protect centromeric cohesin from premature removal (12-14). In addition, Sgo1 has also been shown to promote stable kinetochore-microtubule attachment and sense tension across sister kinetochores (8, 15). Thus, Sgo1 is crucial for mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C),² a large multiprotein ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome (16). APC/C selects substrates for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize specific sequences called APC/C degrons within target proteins (17). Several APC/C degrons have been characterized, including the destruction box (D box) and the Lys-Glu-Asn box (KEN box) (18, 19). The D box, with the consensus amino acid sequence of RXXLXXXN(X indicates any amino acid), are found in many APC/C substrates, including mitotic cyclins and are essential for their ubiquitin-mediated destruction. The KEN box, which contains a consensus KEN motif, is also found in several APC/C substrates and is preferentially but not exclusively recognized by APC/C^Cdh1. When APC/C is active, it directs progression through and exit from mitosis by catalyzing the ubiquitination and timely destruction of mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor securin (16). The APC/C activity needs to be tightly controlled to prevent unscheduled substrate degradation. An important mechanism for APC/C regulation is the spindle checkpoint, which prevents the activation of APC/C and destruction of its substrates in response to kinetochores that have not properly attached to the mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein levels oscillate during the cell cycle (8, 9). In this article we study the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during mitotic exit, and this degradation depends on APC/C^Cdh1. We further show that both KEN and D boxes are required for Sgo1 degradation in vivo and ubiquitination in vitro. Removal of these motifs stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1 protein in human cells does not change the kinetics of chromosome segregation and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes place but is not required for mitotic exit. Finally, we show that Bub1 regulates Sgo1 protein levels through a mechanism that does not involve APC/C-mediated degradation.     

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders

Background

Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders.

Results

First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients’ metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach.

Conclusion

We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

     

MCL1 meets its end during mitotic arrest

Evidence for the destruction of the anti-apoptotic protein MCL1 during prolonged mitotic arrest comes from three papers, one in The EMBO Journal and two in Nature, thus shedding light on the mechanism of apoptosis induction under these conditions.EMBO Rep (2011) advance online publication. doi:10.1038/nature09732EMBO Rep (2011) advance online publication. doi:10.1038/nature09779EMBO Rep (2011) advance online publication. doi:10.1038/emboj.2010.112During mitosis, eukaryotic cells have to properly align their chromosomes. Only after the kinetochore of each chromosome is attached to a polar microtubule can a cell satisfy the ‘spindle assembly checkpoint'', which prevents the mis-segregation of chromosomes. Failure to correctly segregate chromosomes before cell division might contribute to chromosome instability and tumorigenesis. To counteract chromosome aberrations, the cell initiates the apoptotic programme. It has become clear through the use of microtubule-poisoning, chemotherapeutic agents—such as paclitaxel and vincristine—that prolonged activation of the spindle checkpoint can induce mitotic arrest and, subsequently, programmed cell death. The molecular mechanisms responsible for initiating apoptosis during mitotic arrest have remained poorly defined. Two recent papers in Nature (Inuzuka et al, 2011; Wertz et al, 2011) and a report published by the Clarke group last year in The EMBO Journal (Harley et al, 2010) highlight the destruction of MCL1 during prolonged mitotic arrest and shed light on the mechanisms of apoptosis induction.Myeloid cell leukaemia 1 (MCL1) is an anti-apoptotic member of the B-cell lymphoma 2 (BCL2) family of proteins. MCL1, like BCL2 and BCLxL, prevents the downstream activation of BAX and BAK, which are responsible for mitochondrial outer-membrane permeabilization, initiation of the caspase cascade and induction of apoptosis (Youle & Strasser, 2008). Ubiquitination and proteolysis of MCL1 have been reported, but a mechanism for MCL1 degradation following spindle checkpoint activation remains unknown. Now, the studies referenced above suggest that degradation of MCL1 during prolonged mitotic arrest is essential for the induction of apoptosis. Given its prominent role in driving the cell cycle, as well as in safeguarding the fidelity of this process, it is not surprising that the ubiquitin-proteasome system (UPS) has a key role in dictating the activation of the intrinsic apoptotic pathway in cells arrested in mitosis. However, it is surprising that two E3 ubiquitin ligase complexes simultaneously facilitate this degradation event.Harley and colleagues describe the regulation of MCL1 by APC/C^Cdc20(anaphase-promoting complex/cyclosome and its activator Cdc20). This multi-subunit RING E3 ubiquitin ligase is active in mitosis, and ubiquitinates substrates such as securin and cyclin B, thereby allowing progression into anaphase. In their report, Harley and co-workers (2010) demonstrate a Cdk1/cyclin-B-mediated, site-specific phosphorylation (Thr 92 in humans) of MCL1 upon mitotic arrest, followed by its proteolytic destruction by APC/C^Cdc20. Thus, like the sand of an hourglass flipped at each entry into mitosis, the level of MCL1 steadily decreases. If time ‘runs out'' due to a prolonged mitotic arrest (that is, if MCL1 is completely destroyed), then apoptosis is initiated (Fig 1A). Both phosphorylation at Thr 92 and the presence of a conserved destruction or ‘D''-box motif (a characteristic of APC/C substrates) are required for MCL1 proteolysis, although the precise role of phosphorylation in promoting degradation remains unclear.Open in a separate windowFigure 1Two models for proteolytic destruction of MCL1 during mitotic arrest. (A) Schematic illustration of the effects of APC/C^Cdc20 and Cdk1/cyclin B on the degradation of MCL1 during prolonged arrest in mitosis. (B) Schematic illustration of the effects of SCFFbw7, JNK/p38/CKII and Cdk1/cyclin B on MCL1 degradation during mitotic arrest. PP2A is a protein phosphatase that is reported to associate with MCL1. Dashed lines represent inactive processes. Question marks denote unknown mechanisms. APC/C^Cdc20, anaphase-promoting complex/cyclosome and its activator Cdc20; SAC, spindle assembly checkpoint.Interestingly, the stability of MCL1 in asynchronous cells seems to be unaffected when the ability of APC/C^Cdc20 to target MCL1 is compromised by knockdown of Cdc20, or when phosphorylation at Thr 92 is ablated. Although the spindle assembly checkpoint is believed to inhibit APC/C^Cdc20 activity, the degradation of some targets, such as the CDK-inhibitor p21 and cyclin A, is not affected. Consequently, it is possible that MCL1 can be destroyed through Cdc20 during mitotic arrest.More recently, in two reports in Nature (Inuzuka et al, 2011; Wertz et al, 2011), it is shown that MCL1 interacts with another E3 ubiquitin ligase, SCF^Fbxw7. Similarly to the APC/C, the SCF (Skp1/Cul1/F-box protein) is a multi-subunit, RING E3 ubiquitin ligase. The F-box protein provides the specificity for target recognition, often by using specific interaction domains to bind to substrates. In the case of Fbxw7 (also known as Fbw7 and hCdc4), a series of WD40 domains form a pocket that dictates the binding of several substrates. For all known substrates, one or two phosphorylated degradation motifs (phospho-degrons) are recognized by Fbxw7 (Welcker & Clurman, 2008), and MCL1 seems to follow this trend. Briefly, two Fbxw7 degrons—Ser 121/Glu 125 and Ser 159/Thr 163—with different binding affinities were identified in MCL1. Inuzuka and colleagues report that these sites are phosphorylated in a GSK3-dependent manner, supporting a previous report that demonstrated a role for GSK3 in controlling MCL1 degradation (Maurer et al, 2006). They also demonstrate that Fbxw7 affects MCL1 stability during the DNA damage response. Wertz and co-workers provide evidence that, during mitotic arrest, the degrons in MCL1 are instead phosphorylated by JNK, p38 and CKII. Interestingly, when Wertz and colleagues investigated the degradation of MCL1 during mitotic arrest, they discovered a dependence on Fbxw7 similar to that reported for Cdc20 (Fig 1B). Furthermore, a functional Fbxw7–MCL1 interaction was required for the induction of apoptosis in ovarian cancer and T-ALL cell lines treated with microtubule-targeting chemotherapies. This observation presents a dilemma. Which ubiquitin ligase complex—APC/C^Cdc20 or SCF^Fbxw7—targets MCL1 for destruction during mitotic arrest? Do they compete or cooperate?There are several approaches that could be taken to investigate these questions. Perhaps the most promising direction is through understanding the role of various MCL1 phosphorylation events, particularly phosphorylation of Thr 92. The reports collectively demonstrate that Thr 92 and the Fbxw7 degrons are phosphorylated in mitotic cells. It is interesting that Thr 92 phosphorylation is specifically induced at mitosis, and Wertz and colleagues suggest that this event might drive the dissociation of a phosphatase to allow Fbxw7 degron phosphorylation (Fig 1B). However, the results so far are preliminary, and a more complete understanding of the mechanism by which Cdk1/cyclin B phosphorylation of MCL1 promotes proteolysis, and whether this is through Cdc20 and/or Fbxw7, is essential. Although MCL1 degradation after mitotic arrest is unlikely to be associated with the activity of GSK3, is there an induction of GSK3-dependent phosphorylation of MCL1 under other conditions? This important question has been studied previously, but it requires further investigation. Perhaps additional ‘priming'' kinases are involved, as is suspected to be the case for cyclin E and c-Myc, two other substrates of Fbxw7.The concept of a protein being targeted by two ubiquitin ligases is not new. For example, similarly to MCL1, p21 and MLL are targeted by both APC/C^Cdc20 and an SCF complex (SCF^Skp2). Several APC/C^Cdh1 substrates (for example, Cdc25A and claspin) are also degraded via SCFβ^TrCP (Frescas & Pagano, 2008). However, in these instances, APC/C and SCF target the substrates at different phases of the cell cycle. The case of MCL1 is less clear. Wertz and colleagues show that mitotic arrest specifically induces binding of MCL1 to Fbxw7. Conversely, Inuzuka and colleagues provide data suggesting that Fbxw7 loss affects the non-mitotic stability of MCL1. Additionally, in an earlier paper, the Fbxw7 degron was reported to be phosphorylated by GSK3 during cytokine withdrawal (Maurer et al, 2006). Thus, we are left with a picture in which Fbxw7 targets MCL1 during mitotic arrest, but it might also target MCL1 at other points during the cell cycle or in response to external stimuli. With regard to Cdc20-mediated degradation of MCL1, mutation of the D-box seems to stabilize MCL1 only during mitotic arrest, although Cdc20 remains bound to MCL1 in non-mitotic cells. Thus, there might be differences in the conditions for recognition by either Fbxw7 or Cdc20 that merit further investigation. It is also worth mentioning that deubiquitinating enzymes (DUBs) might counteract the activity of Fbxw7, Cdc20, or both. In fact, the DUB USP9X was found to associate with MCL1 (Schwickart et al, 2010).Assuming that both E3 ligases target the same pool of MCL1 at the same time during mitotic arrest, why are there two modes of regulation? It could be that the ligases cooperate to lower MCL1 levels. It is possible that Fbxw7 and Cdc20 together deplete MCL1 to a point at which apoptosis can be initiated; if either ligase is compromised, apoptotic induction is inefficient. Alternatively, there might be a particularly relevant growth condition or cell-type specificity that favours the activity of one complex over the other. For example, it is possible that in tissues that give rise to human cancers harbouring Fbxw7 mutations (for example, T-ALLs or ovarian carcinomas), SCF^Fbxw7 acts as the predominant ligase. Finally, there could be redundancy or competition between the different E3 ligases. Perhaps untransformed cells maintain both systems, to protect against apoptosis evasion in the face of spindle dysfunction. Alternatively, one or both of these systems might be compromised in the cell-culture models. Notably, the situation is further complicated by reports indicating that other ligases seem to affect MCL1 stability: Mule/Huwe1 (Zhong et al, 2005) and SCF^βTrCP (Ding et al, 2007). Silencing of Mule stabilizes MCL1, although Wertz and colleagues did not observe dramatic changes in MCL1 stability after Mule depletion during mitotic arrest. Instead, three groups did not observe stabilization of MCL1 after βTrCP silencing (Wertz et al, 2011; Inuzuka et al, 2011; Dehan et al, 2009). Moreover, the interaction between MCL1 and βTrCP seems to be mediated by BimEL (a βTrCP substrate), as indicated by increased binding under conditions when BimEL is degraded (rather than under conditions when MCL1 is degraded) and by the fact that some BimEL mutants lose their ability to bind to βTrCP, regardless of their binding to MCL1 (Dehan et al, 2009).Although the details of MCL1 regulation at mitotic arrest have only begun to unfold, it is clear that this pathway holds promise for furthering our understanding of the regulation of apoptosis. Microtubule-poisoning agents have historically been reliable chemotherapeutics, so, identifying cellular components that regulate MCL1 degradation during mitotic arrest is not only a way to stratify patients for a positive response to such drugs, but might also lead to the identification of novel targets for pharmacological intervention.     

APC/CCdc20 targets E2F1 for degradation in prometaphase

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase-promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C^Cdc20 is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C^Cdc20 specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.Key words: cell cycle, ubiquitination, E2F1, APC/C, Cdc20, Cdh1     

Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Na?ve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.     

Large area micropatterning of cells on polydimethylsiloxane surfaces

Background

Precise spatial control and patterning of cells is an important area of research with numerous applications in tissue engineering, as well as advancing an understanding of fundamental cellular processes. Poly (dimethyl siloxane) (PDMS) has long been used as a flexible, biocompatible substrate for cell culture with tunable mechanical characteristics. However, fabrication of suitable physico-chemical barriers for cells on PDMS substrates over large areas is still a challenge.

Results

Here, we present an improved technique which integrates photolithography and cell culture on PDMS substrates wherein the barriers to cell adhesion are formed using the photo-activated graft polymerization of polyethylene glycol diacrylate (PEG-DA). PDMS substrates with varying stiffness were prepared by varying the base to crosslinker ratio from 5:1 to 20:1. All substrates show controlled cell attachment confined to fibronectin coated PDMS microchannels with a resistance to non-specific adhesion provided by the covalently immobilized, hydrophilic PEG-DA.

Conclusions

Using photolithography, it is possible to form patterns of high resolution stable at 37°C over 2 weeks, and microstructural complexity over large areas of a few cm². As a robust and scalable patterning method, this technique showing homogenous and stable cell adhesion and growth over macroscales can bring microfabrication a step closer to mass production for biomedical applications.

     

Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2A^Cdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2A^Cdc55 and APC/C^Cdc20 (i.e., a positive feedback loop) that controls APC/C^Cdc20 activity. The circuit could guarantee sustained APC/C^Cdc20 activity after Clb2 starts to be degraded.     

<Emphasis Type="Italic">Sedum</Emphasis> root foraging in layered green roof substrates

Background and aims

Layered profiles of designed soils may provide long-term benefits for green roofs, provided the vegetation can exploit resources in the different layers. We aimed to quantify Sedum root foraging for water and nutrients in designed soils of different texture and layering.

Methods

In a controlled pot experiment we quantified the root foraging ability of the species Sedum album (L.) and S. rupestre (L.) in response to substrate structure (fine, coarse, layered or mixed), vertical fertiliser placement (top or bottom half of pot) and watering (5, 10 or 20 mm week^?1).

Results

Water availability was the main driver of plant growth, followed by substrate structure, while fertiliser placement only had marginal effects on plant growth. Root foraging ability was low to moderate, as also reflected in the low proportion of biomass allocated to roots (5–13%). Increased watering reduced the proportion of root length and root biomass in deeper layers.

Conclusions

Both S. album and S. rupestre had a low ability to exploit water and nutrients by precise root foraging in substrates of different texture and layering. Allocation of biomass to roots was low and showed limited flexibility even under water-deficient conditions.

     

Substrate concentration dependence of voltage and power production characteristics in two-chambered mediator-less microbial fuel cells with acetate and peptone substrates

Objectives

Power production characteristics and substrate concentration dependence of voltage have been investigated together with the determination of kinetic constants in two-chambered mediator-less microbial fuel cells (MFC) for acetate and peptone substrates.

Results

At 500 mg DOC l^?1 (dissolved organic carbon), power densities normalized to the anode surface of 112 mW m^?2 with acetate and 114 mW m^?2 with peptone as electron donor were attained by applying cathodes with a Pt catalyst layer. Related anode surface specific substrate removal rate was 44 g DOC m^?2 h^?1 for acetate and 52 g DOC m^?2 h^?1 for peptone. Substrate concentration dependency of the voltage suggests Monod-like kinetics with extremely low, <1 mg DOC l^?1, half saturation constants and with final DOC concentrations of 6–10 mg l^?1.

Conclusions

Acetate and peptone are equivalent substrates for the exoelectrogenic bacteria both from the point of view of biodegradation kinetics and power production characteristics.

     

Enzymatic synthesis of quercetin oleate esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B

Objectives

To investigate the lipase-catalyzed acylation of quercetin with oleic acid using Candida antarctica lipase B.

Results

Three acylated analogues were produced: quercetin 4′-oleate (C₃₃H₄₂O₈), quercetin 3′,4′-dioleate (C₅₁H₇₄O₉) and quercetin 7,3′,4′-trioleate (C₆₉H₁₀₆O₁₀). Their identities were confirmed with UPLC–ESI–MS and ¹H NMR analyses. The effects of temperature, duration and molar ratio of substrates on the bioconversion yields varied across conditions. The regioselectivity of the acylated quercetin analogues was affected by the molar ratio of substrates. TLC showed the acylated analogues had higher lipophilicity (152% increase) compared to quercetin. Partition coefficient (log P) of quercetin 4′-oleate was higher than those of quercetin and oleic acid. Quercetin 4′-oleate was also stable over 28 days of storage.

Conclusions

Quercetin oleate esters with enhanced lipophilicity can be produced via lipase-catalyzed reaction using C. antarctica lipase B to be used in topical applications.

     

The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

Clb2 and the APC/CCdh1 regulate Swe1 stability

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/C^Cdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5^T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1^mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2^ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5^T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.