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1.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34739034

RESUMO

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endocitose , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Rede trans-Golgi/metabolismo
2.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34694336

RESUMO

Cellular quiescence is a nonproliferative state required for cell survival under stress and during development. In most quiescent cells, proliferation is stopped in a reversible state of low Cdk1 kinase activity; in many organisms, however, quiescent states with high-Cdk1 activity can also be established through still uncharacterized stress or developmental mechanisms. Here, we used a microfluidics approach coupled to phenotypic classification by machine learning to identify stress pathways associated with starvation-triggered high-Cdk1 quiescent states in Saccharomyces cerevisiae. We found that low- and high-Cdk1 quiescent states shared a core of stress-associated processes, such as autophagy, protein aggregation, and mitochondrial up-regulation, but differed in the nuclear accumulation of the stress transcription factors Xbp1, Gln3, and Sfp1. The decision between low- or high-Cdk1 quiescence was controlled by cell cycle-independent accumulation of Xbp1, which acted as a time-delayed integrator of the duration of stress stimuli. Our results show how cell cycle-independent stress-activated factors promote cellular quiescence outside G1/G0.


Assuntos
Ciclo Celular , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Fisiológico , Núcleo Celular/metabolismo , Proliferação de Células , Microfluídica , Fatores de Transcrição/metabolismo
3.
Biochem J ; 478(20): 3791-3805, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34709374

RESUMO

Meiosis facilitates diversity across individuals and serves as a major driver of evolution. However, understanding how meiosis begins is complicated by fundamental differences that exist between sexes and species. Fundamental meiotic research is further hampered by a current lack of human meiotic cells lines. Consequently, much of what we know relies on data from model organisms. However, contextualising findings from yeast, worms, flies and mice can be challenging, due to marked differences in both nomenclature and the relative timing of meiosis. In this review, we set out to combine current knowledge of signalling and transcriptional pathways that control meiosis initiation across the sexes in a variety of organisms. Furthermore, we highlight the emerging links between meiosis initiation and oncogenesis, which might explain the frequent re-expression of normally silent meiotic genes in a variety of human cancers.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Meiose , Oogênese/genética , Espermatogênese/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Humanos , Masculino , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores Sexuais , Transdução de Sinais , Fatores de Tempo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
5.
Chem Commun (Camb) ; 57(81): 10572-10575, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34558573

RESUMO

Insights into intracellular chemistry have remained sparse, but would be impactful for the advancement of biomedicine and bioproduction. A suitable 13C NMR approach provides improvements in sensitivity that make extended reaction networks and assay time windows, previously inaccessible cell densities and relative flux measurements accessible in living cells.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Saccharomyces cerevisiae/citologia , Isótopos de Carbono
6.
FEBS Lett ; 595(18): 2383-2394, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34358326

RESUMO

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/deficiência , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Nucleotídeos/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Domínios Proteicos , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/deficiência
7.
Cells ; 10(7)2021 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-34359924

RESUMO

Complex interactions among DNA and nuclear proteins maintain genome organization and stability. The nuclear proteins, particularly the histones, organize, compact, and preserve the stability of DNA, but also allow its dynamic reorganization whenever the nuclear processes require access to it. Five histone classes exist and they are evolutionarily conserved among eukaryotes. The linker histones are the fifth class and over time, their role in chromatin has been neglected. Linker histones interact with DNA and the other histones and thus sustain genome stability and nuclear organization. Saccharomyces cerevisiae is a brilliant model for studying linker histones as the gene for it is a single-copy and is non-essential. We, therefore, created a linker histone-free yeast strain using a knockout of the relevant gene and traced the way cells age chronologically. Here we present our results demonstrating that the altered chromatin dynamics during the chronological lifespan of the yeast cells with a mutation in ARP4 (the actin-related protein 4) and without the gene HHO1 for the linker histone leads to strong alterations in the gene expression profiles of a subset of genes involved in DNA repair and autophagy. The obtained results further prove that the yeast mutants have reduced survival upon UVA/B irradiation possibly due to the accelerated decompaction of chromatin and impaired proliferation. Our hypothesis posits that the higher-order chromatin structure and the interactions among chromatin proteins are crucial for the maintenance of chromatin organization during chronological aging under optimal and UVA-B stress conditions.


Assuntos
Senescência Celular/efeitos da radiação , Cromatina/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Estresse Fisiológico/efeitos da radiação , Raios Ultravioleta , Ciclo Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Mutação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Fatores de Tempo
8.
FEBS Lett ; 595(17): 2197-2207, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339521

RESUMO

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.


Assuntos
Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/citologia , Vacúolos/metabolismo , Autofagossomos/química , Autofagossomos/metabolismo , Autofagia , Congelamento , Membranas Intracelulares/química , Lipídeos de Membrana/química , Microscopia Eletrônica , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Vacúolos/química
9.
PLoS Genet ; 17(7): e1009560, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34260586

RESUMO

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.


Assuntos
Citoplasma/genética , Proteínas Nucleares/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular , Membrana Celular/metabolismo , Pareamento Cromossômico , Cromossomos Fúngicos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Meiose , Microrganismos Geneticamente Modificados , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
PLoS Comput Biol ; 17(7): e1008525, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34264926

RESUMO

Cells polarize their movement or growth toward external directional cues in many different contexts. For example, budding yeast cells grow toward potential mating partners in response to pheromone gradients. Directed growth is controlled by polarity factors that assemble into clusters at the cell membrane. The clusters assemble, disassemble, and move between different regions of the membrane before eventually forming a stable polarity site directed toward the pheromone source. Pathways that regulate clustering have been identified but the molecular mechanisms that regulate cluster mobility are not well understood. To gain insight into the contribution of chemical noise to cluster behavior we simulated clustering using the reaction-diffusion master equation (RDME) framework to account for molecular-level fluctuations. RDME simulations are a computationally efficient approximation, but their results can diverge from the underlying microscopic dynamics. We implemented novel concentration-dependent rate constants that improved the accuracy of RDME-based simulations, allowing us to efficiently investigate how cluster dynamics might be regulated. Molecular noise was effective in relocating clusters when the clusters contained low numbers of limiting polarity factors, and when Cdc42, the central polarity regulator, exhibited short dwell times at the polarity site. Cluster stabilization occurred when abundances or binding rates were altered to either lengthen dwell times or increase the number of polarity molecules in the cluster. We validated key results using full 3D particle-based simulations. Understanding the mechanisms cells use to regulate the dynamics of polarity clusters should provide insights into how cells dynamically track external directional cues.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Simulação por Computador , Modelos Biológicos , Algoritmos , Membrana Celular/fisiologia , Biologia Computacional , Difusão , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Processos Estocásticos
11.
Commun Biol ; 4(1): 822, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193958

RESUMO

Stochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Análise por Conglomerados , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Guanina/metabolismo , Guanina/farmacologia , Ácido Micofenólico/metabolismo , Ácido Micofenólico/farmacologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Análise de Sequência de RNA/métodos , Transcriptoma/efeitos dos fármacos
12.
J Cell Biol ; 220(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34283201

RESUMO

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)-endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteróis/biossíntese , Transporte Biológico , Membrana Celular/metabolismo , Endocitose , Saccharomyces cerevisiae/citologia
13.
Biochem Biophys Res Commun ; 570: 125-130, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34280615

RESUMO

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3' poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3'-5' RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.


Assuntos
Carbono/farmacologia , Fermentação , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mutação/genética , Fosforilação/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos
14.
PLoS Comput Biol ; 17(7): e1009188, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34297727

RESUMO

Cellular RNA levels typically fluctuate and are influenced by different transcription rates and RNA degradation rates. However, the understanding of the fundamental relationships between RNA abundance, environmental stimuli, RNA activities, and RNA age distributions is incomplete. Furthermore, the rates of RNA degradation and transcription are difficult to measure in transcriptomic experiments in living organisms, especially in studies involving humans. A model based on activity demands and RNA age was developed to explore the mechanisms of RNA level fluctuations. Using single-cell time-series gene expression experimental data, we assessed the transcription rates, RNA degradation rates, RNA life spans, RNA demand, accumulated transcription levels, and accumulated RNA degradation levels. This model could also predict RNA levels under simulation backgrounds, such as stimuli that induce regular oscillations in RNA abundance, stable RNA levels over time that result from long-term shortage of total RNA activity or from uncontrollable transcription, and relationships between RNA/protein levels and metabolic rates. This information contributes to existing knowledge.


Assuntos
Modelos Biológicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Biologia Computacional , Simulação por Computador , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única , Transcrição Genética , Transcriptoma
15.
Artigo em Inglês | MEDLINE | ID: mdl-34062255

RESUMO

SBF (Swi4/Swi6 Binding Factor) complex is a crucial regulator of G1/S transition in Saccharomyces cerevisiae. Here, we show that SBF complex is required for myriocin resistance, an inhibitor of sphingolipid synthesis. This phenotype was not shared with MBF complex mutants nor with deletion of the Swi4p downstream targets, CLN1/CLN2. Based on data mining results, we selected putative Swi4p targets related to sphingolipid metabolism and studied their gene transcription as well as metabolite levels during progression of the cell cycle. Genes which encode key enzymes for the synthesis of long chain bases (LCBs) and ceramides were periodically transcribed during the mitotic cell cycle, having a peak at G1/S, and required SWI4 for full transcription at this stage. In addition, HPLC-MS/MS data indicated that swi4Δ cells have decreased levels of sphingolipids during progression of the cell cycle, particularly, dihydrosphingosine (DHS), C24-phytoceramides and C24-inositolphosphoryl ceramide (IPC) while it had increased levels of mannosylinositol phosphorylceramide (MIPC). Furthermore, we demonstrated that both inhibition of de novo sphingolipid synthesis by myriocin or SWI4 deletion caused partial arrest at the G2/M phase. Importantly, our lipidomic data demonstrated that the sphingolipid profile of WT cells treated with myriocin resembled that of swi4Δ cells, with lower levels of DHS, IPC and higher levels of MIPC. Taken together, these results show that SBF complex plays an essential role in the regulation of sphingolipid homeostasis, which reflects in the correct progression through the G2/M phase of the cell cycle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/biossíntese , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Mitose/genética , Saccharomyces cerevisiae/genética
16.
Methods Mol Biol ; 2277: 69-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080145

RESUMO

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Equorina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Descoberta de Drogas/métodos , Humanos , Ácido Láctico/farmacologia , Mitocôndrias/metabolismo , Mitoxantrona/farmacologia , Saccharomyces cerevisiae/citologia
17.
Sci Rep ; 11(1): 12819, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140587

RESUMO

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.


Assuntos
Senescência Celular , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Corantes Fluorescentes/metabolismo , Temperatura Alta , Espaço Intracelular/metabolismo , Agregados Proteicos , Transporte Proteico , Saccharomyces cerevisiae/crescimento & desenvolvimento
18.
Ultrason Sonochem ; 76: 105624, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34126524

RESUMO

In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca2+ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.


Assuntos
Reatores Biológicos , Cálcio/metabolismo , Etanol/metabolismo , Fermentação , Espaço Intracelular/metabolismo , Saccharomyces cerevisiae/metabolismo , Sonicação , Glicólise , Saccharomyces cerevisiae/citologia
19.
PLoS One ; 16(6): e0248382, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34111115

RESUMO

The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos/microbiologia , Fluorometria , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento
20.
PLoS Genet ; 17(5): e1009592, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34033659

RESUMO

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.


Assuntos
Anáfase , Aurora Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Segregação de Cromossomos , Cinetocoros/química , Cinetocoros/efeitos dos fármacos , Viabilidade Microbiana/genética , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/efeitos dos fármacos , Fatores de Tempo
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