RESUMO
Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells characterized by high intratumoral variation. We use highly multiplexed tissue imaging, 3D reconstruction, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. Quantitation of these features in high-plex marker space reveals recurrent transitions from one tumor morphology to the next, some of which are coincident with long-range gradients in the expression of oncogenes and epigenetic regulators. At the tumor invasive margin, where tumor, normal, and immune cells compete, T cell suppression involves multiple cell types and 3D imaging shows that seemingly localized 2D features such as tertiary lymphoid structures are commonly interconnected and have graded molecular properties. Thus, while cancer genetics emphasizes the importance of discrete changes in tumor state, whole-specimen imaging reveals large-scale morphological and molecular gradients analogous to those in developing tissues.
Assuntos
Adenocarcinoma , Neoplasias Colorretais , Humanos , Adenocarcinoma/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Processamento de Imagem Assistida por Computador , Oncogenes , Microambiente TumoralRESUMO
Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.
Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Animais , Morte Celular , Sobrevivência Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Mamíferos/genética , Camundongos Endogâmicos C57BL , RNA/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.
Assuntos
Junções Célula-Matriz/metabolismo , Morfogênese , Animais , Membrana Basal/metabolismo , Adesão Celular , Divisão Celular , Movimento Celular , Rastreamento de Células , Embrião de Mamíferos/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Integrinas/metabolismo , Camundongos , Modelos Biológicos , Glândulas Salivares/citologia , Glândulas Salivares/embriologia , Glândulas Salivares/metabolismo , Transcriptoma/genéticaRESUMO
Exosomes are small, single-membrane, secreted organelles of â¼30 to â¼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.
Assuntos
Exossomos/metabolismo , Animais , Transporte Biológico , Exossomos/imunologia , Exossomos/fisiologia , Exossomos/ultraestrutura , Matriz Extracelular/metabolismo , Humanos , Neoplasias , Doenças Neurodegenerativas , Multimerização Proteica , Transdução de SinaisRESUMO
Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.
Assuntos
Eucariotos/genética , Transferência Genética Horizontal/genética , Óperon/genética , Bactérias/genética , Escherichia coli/genética , Células Eucarióticas , Evolução Molecular , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Genoma Bacteriano/genética , Genoma Fúngico/genética , Saccharomycetales/genética , Sideróforos/genéticaRESUMO
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genéticaRESUMO
DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.
Assuntos
Saccharomyces cerevisiae , Transcrição Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , DNA Topoisomerases Tipo II/genética , DNA , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , DNA Topoisomerases Tipo I/metabolismoRESUMO
mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular , Citoplasma/genética , Citoplasma/metabolismo , Regulação Fúngica da Expressão Gênica , Carioferinas/genética , Carioferinas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/classificação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
To fulfill their actual cellular role, individual microtubules become functionally specialized through a broad range of mechanisms. The 'search and capture' model posits that microtubule dynamics and functions are specified by cellular targets that they capture (i.e., a posteriori), independently of the microtubule-organizing center (MTOC) they emerge from. However, work in budding yeast indicates that MTOCs may impart a functional identity to the microtubules they nucleate, a priori. Key effectors in this process are microtubule plus-end tracking proteins (+TIPs), which track microtubule tips to regulate their dynamics and facilitate their targeted interactions. In this review, we discuss potential mechanisms of a priori microtubule specialization, focusing on recent findings indicating that +TIP networks may undergo liquid biomolecular condensation in different cell types.
Assuntos
Proteínas Associadas aos Microtúbulos , Microtúbulos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMO
The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.
Assuntos
Meiose , Proteínas de Saccharomyces cerevisiae , Nucleossomos , Microscopia Crioeletrônica , Filogenia , Saccharomyces cerevisiae/genética , DNA , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.
RESUMO
DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de TempoRESUMO
The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
Assuntos
Replicação do DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Origem de Replicação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Across eukaryotes, disruption of DNA replication causes an S phase checkpoint response, which regulates multiple processes, including inhibition of replication initiation and fork stabilization. How these events are coordinated remains poorly understood. Here, we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to forkhead-associated (FHA) interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally, we show that a Cdc45 mutation found in patients with Meier-Gorlin syndrome disrupts the functional interaction with Rad53 in yeast. Together, we present a single mechanism by which a checkpoint kinase targets replication initiation and elongation complexes, which may be relevant to human disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Microtia Congênita/enzimologia , Microtia Congênita/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Humanos , Micrognatismo/enzimologia , Micrognatismo/genética , Mutação , Proteínas Nucleares/genética , Patela/anormalidades , Patela/enzimologia , Fosforilação , Ligação Proteica , Pontos de Checagem da Fase S do Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and ß2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.
Assuntos
Complexo 2 de Proteínas Adaptadoras , Chaperonas Moleculares , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Ligação Proteica , Endocitose/fisiologia , Transporte ProteicoRESUMO
Progerin causes Hutchinson-Gilford progeria syndrome (HGPS), but how progerin accelerates aging is still an interesting question. Here, we provide evidence linking nuclear envelope (NE) budding and accelerated aging. Mechanistically, progerin disrupts nuclear lamina to induce NE budding in concert with lamin A/C, resulting in transport of chromatin into the cytoplasm where it is removed via autophagy, whereas emerin antagonizes this process. Primary cells from both HGPS patients and mouse models express progerin and display NE budding and chromatin loss, and ectopically expressing progerin in cells can mimic this process. More excitingly, we screen a NE budding inhibitor chaetocin by high-throughput screening, which can dramatically sequester progerin from the NE and prevent this NE budding through sustaining ERK1/2 activation. Chaetocin alleviates NE budding-induced chromatin loss and ameliorates HGPS defects in cells and mice and significantly extends lifespan of HGPS mice. Collectively, we propose that progerin-induced NE budding participates in the induction of progeria, highlight the roles of chaetocin and sustained ERK1/2 activation in anti-aging, and provide a distinct avenue for treating HGPS.
Assuntos
Lamina Tipo A , Membrana Nuclear , Proteínas Nucleares , Progéria , Progéria/metabolismo , Progéria/tratamento farmacológico , Progéria/patologia , Progéria/genética , Animais , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos , Humanos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Envelhecimento/metabolismo , Envelhecimento/efeitos dos fármacos , Cromatina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Modelos Animais de Doenças , Autofagia/efeitos dos fármacosRESUMO
Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.
Assuntos
Membrana Celular , Ferroptose , Corantes Fluorescentes , Microscopia de Fluorescência , Membrana Nuclear , Ferroptose/fisiologia , Humanos , Corantes Fluorescentes/química , Membrana Celular/metabolismo , Membrana Nuclear/metabolismo , Microscopia de Fluorescência/métodos , Exocitose/fisiologia , Células HeLaRESUMO
The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.
Assuntos
Lipídeos de Membrana , Organelas , Transporte Biológico , Homeostase , Metabolismo dos Lipídeos/genética , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Organelas/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.
Assuntos
Proteínas 14-3-3 , Segregação de Cromossomos , Cinetocoros , Mitose , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cinetocoros/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Microtúbulos/metabolismo , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/genéticaRESUMO
Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.