Xrn1 Exoribonuclease-An Intrinsic Marker of Yeast Population Growth.

BACKGROUND: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown. METHODS: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δxrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy. RESULTS: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy. CONCLUSIONS: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy.

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes - the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines - the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.

Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment.

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

ERK2 signaling regulates cell-cell adhesion of epithelial cells and enhances growth factor-induced cell scattering.

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.

Yeast stress granules at a glance.

The formation of stress granules (SGs), membrane-less organelles that are composed of mainly messenger ribonucleoprotein assemblies, is the result of a conserved evolutionary strategy to cellular stress. During their formation, which is triggered by robust environmental stress, SGs sequester translationally inactive mRNA molecules, which are either forwarded for further processing elsewhere or stored during a period of stress within SGs. Removal of mRNA molecules from active translation and their sequestration in SGs allows preferential translation of stress response transcripts. By affecting the specificity of mRNA translation, mRNA localization and stability, SGs are involved in the overall cellular reprogramming during periods of environmental stress and viral infection. Over the past two decades, we have learned which processes drive SGs assembly, how their composition varies under stress, and how they co-exist with other subcellular organelles. Yeast as a model has been instrumental in our understanding of SG biology. Despite the specific differences between the SGs of yeast and mammals, yeast have been shown to be a valuable tool to the study of SGs in translation-related stress response. This review summarizes the data surrounding SGs that are formed under different stress conditions in Saccharomyces cerevisiae and other yeast species. It offers a comprehensive and up-to-date view on these still somewhat mysterious entities.

Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.

Quantitative Phase Imaging of Spreading Fibroblasts Identifies the Role of Focal Adhesion Kinase in the Stabilization of the Cell Rear.

Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.

Cellular sequestrases maintain basal Hsp70 capacity ensuring balanced proteostasis.

Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.

A prion-like domain in Hsp42 drives chaperone-facilitated aggregation of misfolded proteins.

Chaperones with aggregase activity promote and organize the aggregation of misfolded proteins and their deposition at specific intracellular sites. This activity represents a novel cytoprotective strategy of protein quality control systems; however, little is known about its mechanism. In yeast, the small heat shock protein Hsp42 orchestrates the stress-induced sequestration of misfolded proteins into cytosolic aggregates (CytoQ). In this study, we show that Hsp42 harbors a prion-like domain (PrLD) and a canonical intrinsically disordered domain (IDD) that act coordinately to promote and control protein aggregation. Hsp42 PrLD is essential for CytoQ formation and is bifunctional, mediating self-association as well as binding to misfolded proteins. Hsp42 IDD confines chaperone and aggregase activity and affects CytoQ numbers and stability in vivo. Hsp42 PrLD and IDD are both crucial for cellular fitness during heat stress, demonstrating the need for sequestering misfolded proteins in a regulated manner.

Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding.

Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation.

Systemic control of protein synthesis through sequestration of translation and ribosome biogenesis factors during severe heat stress.

Environmental stress causes the sequestration of proteins into insoluble deposits including cytoplasmic stress granules (SGs), containing mRNA and a variety of translation factors. Here we systematically identified proteins sequestered in Saccharomyces cerevisiae at 46 °C by a SG co-localization screen and proteomic analysis of insoluble protein fractions. We identified novel SG components including essential aminoacyl-tRNA synthetases. Moreover, we discovered nucleus-associated deposits containing ribosome biogenesis factors. Our study suggests downregulation of cytosolic protein synthesis and nuclear ribosome production at multiple levels through heat shock induced protein sequestrations.

Evolutionarily conserved 5'-3' exoribonuclease Xrn1 accumulates at plasma membrane-associated eisosomes in post-diauxic yeast.

Regulation of gene expression on the level of translation and mRNA turnover is widely conserved evolutionarily. We have found that the main mRNA decay enzyme, exoribonuclease Xrn1, accumulates at the plasma membrane-associated eisosomes after glucose exhaustion in a culture of the yeast S. cerevisiae. Eisosomal localization of Xrn1 is not achieved in cells lacking the main component of eisosomes, Pil1, or Sur7, the protein accumulating at the membrane compartment of Can1 (MCC) - the eisosome-organized plasma membrane microdomain. In contrast to the conditions of diauxic shift, when Xrn1 accumulates in processing bodies (P-bodies), or acute heat stress, in which these cytosolic accumulations of Xrn1 associate with eIF3a/Rpg1-containing stress granules, Xrn1 is not accompanied by other mRNA-decay machinery components when it accumulates at eisosomes in post-diauxic cells. It is important that Xrn1 is released from eisosomes after addition of fermentable substrate. We suggest that this spatial segregation of Xrn1 from the rest of the mRNA-decay machinery reflects a general regulatory mechanism, in which the key enzyme is kept separate from the rest of mRNA decay factors in resting cells but ready for immediate use when fermentable nutrients emerge and appropriate metabolism reprogramming is required. In particular, the localization of Xrn1 to the eisosome, together with previously published data, accents the relevance of this plasma membrane-associated compartment as a multipotent regulatory site.

Mmi1, the yeast homologue of mammalian TCTP, associates with stress granules in heat-shocked cells and modulates proteasome activity.

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.

Heat shock-induced accumulation of translation elongation and termination factors precedes assembly of stress granules in S. cerevisiae.

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Robust heat shock induces eIF2alpha-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae.

Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2alpha/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Deltalsm4DeltaC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2alpha became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2alpha. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division.

The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11+ and cbf12+, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11+ and cbf12+ are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTGA/GGAA in vitro. The deletion of cbf11+ is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.