Angelika Amon

A Mechanism for Coupling Exit from Mitosis to Partitioning of the Nucleus

Exit from mitosis must not occur prior to partitioning of chromosomes between daughter cells. We find that the GTP binding protein Tem1, a regulator of mitotic exit, is present on the spindle pole body that migrates into the bud during S phase and mitosis. Tem1s exchange factor, Lte1, localizes to the bud. Thus, Tem1 and Lte1 are present in the same cellular compartment (the bud) only after the nucleus enters the bud during nuclear division. We also find that the presence of Tem1 and Lte1 in the bud is required for mitotic exit. Our results suggest that the spatial segregation of Tem1 and Lte1 ensures that exit from mitosis only occurs after the genetic material is partitioned between mother and daughter cell.

The FK506 Binding Protein Fpr3 Counteracts Protein Phosphatase 1 to Maintain Meiotic Recombination Checkpoint Activity

The meiotic recombination checkpoint delays gamete precursors in G2 until DNA breaks created during recombination are repaired and chromosome structure has been restored. Here, we show that the FK506 binding protein Fpr3 prevents premature adaptation to damage and thus serves to maintain recombination checkpoint activity. Impaired checkpoint function is observed both in cells lacking FPR3 and in cells treated with rapamycin, a small molecule inhibitor that binds to the proline isomerase (PPIase) domain of Fpr3. FPR3 functions in the checkpoint through controlling protein phosphatase 1 (PP1). Fpr3 interacts with PP1 through its PPIase domain, regulates PP1 localization, and counteracts the activity of PP1 in vivo. Our findings define a branch of the recombination checkpoint involved in the adaptation to persistent chromosomal damage and a critical function for FK506 binding proteins during meiosis.

The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation.

During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation. Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally. We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events. Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle. Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.

The Replication Fork Block Protein Fob1 Functions as a Negative Regulator of the FEAR Network

BACKGROUND The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase. RESULTS Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1. CONCLUSIONS Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.

Regulated Formation of an Amyloid-like Translational Repressor Governs Gametogenesis

Message-specific translational control is required for gametogenesis. In yeast, the RNA-binding protein Rim4 mediates translational repression of numerous mRNAs, including the B-type cyclin CLB3, which is essential for establishing the meiotic chromosome segregation pattern. Here, we show that Rim4 forms amyloid-like aggregates and that it is the amyloid-like form of Rim4 that is the active, translationally repressive form of the protein. Our data further show that Rim4 aggregation is a developmentally regulated process. Starvation induces the conversion of monomeric Rim4 into amyloid-like aggregates, thereby activating the protein to bring about repression of translation. At the onset of meiosis II, Rim4 aggregates are abruptly degraded allowing translation to commence. Although amyloids are best known for their role in the etiology of diseases such as Alzheimers, Parkinsons, and diabetes by forming toxic protein aggregates, our findings show that cells can utilize amyloid-like protein aggregates to function as central regulators of gametogenesis.

The FEAR network

Mitosis is governed by the oscillation of cyclin dependent kinase (CDK) activity and ubiquitin-dependent proteolysis. Entry into mitosis is initiated by mitotic cyclin-CDK activation. Anaphase onset occurs upon activation of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that promotes the destruction of the anaphase inhibitor Securin. Destruction of Securin initiates chromosome segregation by activation of the protease Separase, allowing it to cleave a subunit of the cohesin complexes that hold the duplicated sister chromatids together. Upon completion of nuclear division cells exit from mitosis, a process defined by the inactivation of CDKs, disassembly of the mitotic spindle, and cytokinesis. In the budding yeast S. cerevisiae, a signaling network known as the FEAR network is critical to ensure accurate anaphase chromosome segregation and the integration of this process with other anaphase events. Here, we summarize what is known about the regulation and function of the FEAR network in budding yeast and discuss the potential for conserved FEAR network functions in other eukaryotes.

MitoCPR-A surveillance pathway that protects mitochondria in response to protein import stress.

The mitoCPR unclogs mitochondria The import of proteins into mitochondria is essential for cell viability. How cells respond when mitochondrial protein import is impaired is poorly understood. Weidberg and Amon showed that upon mitochondrial import stress, yeast cells mounted a response known as the mitoCPR. mitoCPR was activated when mitochondrial protein import was impaired and unimported precursors accumulated on the organelles surface. mitoCPR restored mitochondrial functions by clearing stalled proteins from the import channels. It did this by inducing expression of Cis1, which recruited the adenosine triphosphatase Msp1 to import channels to remove unimported precursors and target them for degradation by the proteasome. Science, this issue p. eaan4146 MitoCPR triggers the removal of unimported mitochondrial precursors and restores function during protein import stress in yeast. INTRODUCTION Mitochondria provide cells with energy and numerous essential metabolites such as lipids, amino acids, iron sulfur clusters, and heme. All mitochondrial functions rely on import of proteins into the organelle because the mitochondrial proteome is almost exclusively encoded by nuclear genes. Given the central importance of mitochondria for cell viability, it is not surprising that cells mount a nuclear response when mitochondrial functions are compromised. These mitochondria-to-nucleus signaling pathways include the mtUPR (mitochondrial unfolded protein response), which triggers expression of mitochondrial chaperones when mitochondrial protein folding is defective, and the UPRam (unfolded protein response activated by mistargeting of proteins) and mPOS (mitochondrial precursor over-accumulation stress) pathways, which reduce translation and induce degradation of unimported proteins in the cytosol when mitochondrial import is impaired. Even though mitochondrial import is central to all mitochondrial functions, no response to protein import defects had been described that protects mitochondria during this stress. RATIONALE To determine how cells respond to defects in mitochondrial protein import, we first developed a system in budding yeast with which to specifically inhibit this process. We found that overexpression of proteins that rely on a bipartite signal sequence for their mitochondrial localization inhibited mitochondrial import and led to the accumulation of mitochondrial precursors. Protease protection and carbonate extraction assays that were performed on isolated mitochondria revealed that these unimported proteins accumulated on the mitochondrial surface and in the import channel known as the translocase. RESULTS Having developed a system that allowed us to specifically inhibit mitochondrial protein import, we examined the cellular response to this defect. Transcriptome analysis of cells overexpressing bipartite signal–containing proteins identified a gene expression pattern related to the multi-drug resistance response. We termed this response mitochondrial compromised protein import response (mitoCPR). mitoCPR was triggered by protein import defects but not other mitochondrial deficiencies, such as respiratory failure, and was mediated by the transcription factor Pdr3. Our analyses further showed that mitoCPR was critical for the protection of mitochondria during import stress. Cells lacking PDR3 did not mount a mitoCPR during import stress and accumulated higher levels of unimported proteins on the organelle surface as compared with those of wild-type cells. Consequently, pdr3Δ cells exhibited decreased respiratory function and loss of mitochondrial DNA when mitochondrial import was restored. Our results also shed light on the mechanism by which mitoCPR protected mitochondria. Upon mitochondrial import stress, Pdr3 induced expression of Cis1. Coimmunoprecipitation analyses showed that Cis1 recruited the AAA+ adenosine triphosphatase Msp1 to the translocase by binding to the translocase receptor Tom70. There, the two proteins mediated the clearance and proteasomal degradation of proteins that failed to be imported into mitochondria. CONCLUSION We discovered a mitochondrial import surveillance mechanism in budding yeast. This surveillance mechanism, mitoCPR, is activated when mitochondrial import is stalled in order to induce the removal of mitochondrial proteins accumulating on the mitochondrial surface. Clearance of precursors is critical for maintaining mitochondrial functions during import stress. We propose that mitoCPR could be especially important when the import machinery is overwhelmed, as may occur in situations that require the rapid expansion of the mitochondrial compartment. MitoCPR protects mitochondria during import stress. (Left) Mitochondrial protein import deficiency leads to the accumulation of mitochondrial proteins on the organelle’s surface and in the translocases. (Right) Pdr3 induces CIS1 expression. Cis1 binds to the mitochondrial import receptor Tom70 and recruits Msp1 to mediate clearance of unimported precursors from the mitochondrial surface and their proteasomal degradation. This protects mitochondrial functions during import stress. ILLUSTRATION: ELLA MARU STUDIO Mitochondrial functions are essential for cell viability and rely on protein import into the organelle. Various disease and stress conditions can lead to mitochondrial import defects. We found that inhibition of mitochondrial import in budding yeast activated a surveillance mechanism, mitoCPR, that improved mitochondrial import and protected mitochondria during import stress. mitoCPR induced expression of Cis1, which associated with the mitochondrial translocase to reduce the accumulation of mitochondrial precursor proteins at the mitochondrial translocase. Clearance of precursor proteins depended on the Cis1-interacting AAA+ adenosine triphosphatase Msp1 and the proteasome, suggesting that Cis1 facilitates degradation of unimported proteins. mitoCPR was required for maintaining mitochondrial functions when protein import was compromised, demonstrating the importance of mitoCPR in protecting the mitochondrial compartment.

Aneuploid Cell Survival Relies upon Sphingolipid Homeostasis

Aneuploidy, a hallmark of cancer cells, poses an appealing opportunity for cancer treatment and prevention strategies. Using a cell-based screen to identify small molecules that could selectively kill aneuploid cells, we identified the compound N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide monohydrochloride (DL-PDMP), an antagonist of UDP-glucose ceramide glucosyltransferase. DL-PDMP selectively inhibited proliferation of aneuploid primary mouse embryonic fibroblasts and aneuploid colorectal cancer cells. Its selective cytotoxic effects were based on further accentuating the elevated levels of ceramide, which characterize aneuploid cells, leading to increased apoptosis. We observed that DL-PDMP could also enhance the cytotoxic effects of paclitaxel, a standard-of-care chemotherapeutic agent that causes aneuploidy, in human colon cancer and mouse lymphoma cells. Our results offer pharmacologic evidence that the aneuploid state in cancer cells can be targeted selectively for therapeutic purposes, or for reducing the toxicity of taxane-based drug regimens. Cancer Res; 77(19); 5272-86. ©2017 AACR.

Deregulation of the G1/S-phase transition is the proximal cause of mortality in old yeast mother cells

Budding yeast cells produce a finite number of daughter cells before they die. Why old yeast cells stop dividing and die is unclear. We found that age-induced accumulation of the G1/S-phase inhibitor Whi5 and defects in G1/S cyclin transcription cause cell cycle delays and genomic instability that result in cell death. We further identified extrachromosomal rDNA (ribosomal DNA) circles (ERCs) to cause the G1/S cyclin expression defect in old cells. Spontaneous segregation of Whi5 and ERCs into daughter cells rejuvenates old mothers, but daughters that inherit these aging factors die rapidly. Our results identify deregulation of the G1/S-phase transition as the proximal cause of age-induced proliferation decline and cell death in budding yeast.

Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture

Much of our understanding of chromosome segregation is based on cell culture systems. Here, we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems, we show that tissue architecture, specifically integrin function, is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments, and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover, our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue.