RESUMEN
For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.
Asunto(s)
Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citología , Cromosomas Fúngicos/genética , Microscopía Fluorescente/métodos , Saccharomycetales/genética , Saccharomycetales/citologíaRESUMEN
Autophagy is essential for maintaining glucose homeostasis. However, the mechanism by which cells sense and respond to glucose starvation to induce autophagy remains incomplete. Here, we show that calcium serves as a fundamental triggering signal that connects environmental sensing to the formation of the autophagy initiation complex during glucose starvation. Mechanistically, glucose starvation instigates the release of vacuolar calcium into the cytoplasm, thus triggering the activation of Rck2 kinase. In turn, Rck2-mediated Atg11 phosphorylation enhances Atg11 interactions with Bmh1/2 bound to the Snf1-Sip1-Snf4 complex, leading to recruitment of vacuolar membrane-localized Snf1 to the PAS and subsequent Atg1 activation, thereby initiating autophagy. We also identified Glc7, a protein phosphatase-1, as a critical regulator of the association between Bmh1/2 and the Snf1 complex. We thus propose that calcium-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy by controlling Snf1-mediated Atg1 activation in response to glucose starvation.
Asunto(s)
Autofagia , Glucosa , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Calcio/metabolismo , Glucosa/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Vacuolas/genéticaRESUMEN
Oxygen levels vary in the environment. Oxygen availability has a major effect on almost all organisms, and oxygen is far more than a substrate for energy production. However, less is known about related biological processes under hypoxic conditions and about the adaptations to changing oxygen concentrations. The yeast Saccharomyces cerevisiae can adapt its metabolism for growth under different oxygen concentrations and can grow even under anaerobic conditions. Therefore, we developed a microfluidic device that can generate serial, accurately controlled oxygen concentrations for single-cell studies of multiple yeast strains. This device can construct a broad range of oxygen concentrations, [O2] through on-chip gas-mixing channels from two gases fed to the inlets. Gas diffusion through thin polydimethylsiloxane (PDMS) can lead to the equilibration of [O2] in the medium in the cell culture layer under gas cover regions within 2 min. Here, we established six different and stable [O2] varying between ~0.1 and 20.9% in the corresponding layers of the device designed for multiple parallel single-cell culture of four different yeast strains. Using this device, the dynamic responses of different yeast transcription factors and metabolism-related proteins were studied when the [O2] decreased from 20.9% to serial hypoxic concentrations. We showed that different hypoxic conditions induced varying degrees of transcription factor responses and changes in respiratory metabolism levels. This device can also be used in studies of the aging and physiology of yeast under different oxygen conditions and can provide new insights into the relationship between oxygen and organisms. Integration, innovation and insight: Most living cells are sensitive to the oxygen concentration because they depend on oxygen for survival and proper cellular functions. Here, a composite microfluidic device was designed for yeast single-cell studies at a series of accurately controlled oxygen concentrations. Using this device, we studied the dynamic responses of various transcription factors and proteins to changes in the oxygen concentration. This study is the first to examine protein dynamics and temporal behaviors under different hypoxic conditions at the single yeast cell level, which may provide insights into the processes involved in yeast and even mammalian cells. This device also provides a base model that can be extended to oxygen-related biology and can acquire more information about the complex networks of organisms.
Asunto(s)
Oxígeno , Saccharomyces cerevisiae , Análisis de la Célula Individual , Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Dimetilpolisiloxanos/química , Dispositivos Laboratorio en un Chip , Proteínas de Saccharomyces cerevisiae/metabolismo , Diseño de Equipo , Técnicas Analíticas Microfluídicas/instrumentación , MicrofluídicaRESUMEN
Cell division without cell separation produces multicellular clusters in budding yeast. Two fundamental characteristics of these clusters are their size (the number of cells per cluster) and cellular composition: the fractions of cells with different phenotypes. Using cells as nodes and links between mother and daughter cells as edges, we model cluster growth and breakage by varying three parameters: the cell division rate, the rate at which intercellular connections break, and the kissing number (the maximum number of connections to one cell). We find that the kissing number sets the maximum possible cluster size. Below this limit, the ratio of the cell division rate to the connection breaking rate determines the cluster size. If links have a constant probability of breaking per unit time, the probability that a link survives decreases exponentially with its age. Modeling this behavior recapitulates experimental data. We then use this framework to examine synthetic, differentiating clusters with two cell types, faster-growing germ cells and their somatic derivatives. The fraction of clusters that contain both cell types increases as either of two parameters increase: the kissing number and difference between the growth rate of germ and somatic cells. In a population of clusters, the variation in cellular composition is inversely correlated (r2 = 0.87) with the average fraction of somatic cells in clusters. Our results show how a small number of cellular features can control the phenotypes of multicellular clusters that were potentially the ancestors of more complex forms of multicellular development, organization, and reproduction.
Asunto(s)
Modelos Biológicos , Fenotipo , División Celular/fisiología , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/citologíaRESUMEN
Cells can respond and adapt to complex forms of environmental change. Budding yeast is widely used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a microheater (using liquid metal) and a thermocouple (liquid metal vs. conductive PDMS) on a chip. Three forms of temperature changes (step, gradient, and periodical oscillations) were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37 °C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated.
Asunto(s)
Respuesta al Choque Térmico , Técnicas Analíticas Microfluídicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Análisis de la Célula Individual , Temperatura , Análisis de la Célula Individual/instrumentación , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/citología , Dispositivos Laboratorio en un Chip , Dimetilpolisiloxanos/química , Diseño de Equipo , Factores de TranscripciónRESUMEN
QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos , Ingeniería Metabólica , Saccharomyces cerevisiae , Saponinas , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Vías Biosintéticas/genética , Diseño de Fármacos , Enzimas/genética , Enzimas/metabolismo , Ingeniería Metabólica/métodos , Plantas/enzimología , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saponinas/biosíntesis , Saponinas/química , Saponinas/genética , Saponinas/metabolismo , Relación Estructura-ActividadRESUMEN
Size-based particle filtration has become indispensable in numerous biomedical and environmental applications. In this study, bioinspired by the filter-feeding mechanism (lobe filtration) of manta rays, we designed a U-shaped biomimetic gill rake filter that combined lobe filtration and Dean flow to filter monodisperse suspensions, bi-disperse suspensions and yeast cells. Compared with other equipment using the inertial focusing technology, our equipment can perform high-throughput (up to 8 mL min-1) and high-efficiency filtration of particles (maximum filtration efficiencies of 96.08% and 97.14% for 10 and 15 µm monodisperse suspensions at the optimum flow rate of 6 mL min-1). The complex velocity field of the micro-fluidic flow within the filter is numerically simulated, and in combination with experiments, a threshold for the flow rate is identified. When the inlet flow rate exceeds the threshold value, the efficiency of particle filtration is increased rapidly. Afterwards, by analysing the filtration mechanism, we develop three novel filtration processes. The equilibrium positions of the particles and yeast cells in the main channel are close to the outer wall at high flow rate, which diminishes the likelihood of particles and yeast cells entering the side channel. This configuration establishes a self-cleaning mechanism, ensuring prolonged and efficient operation of the filter with high-throughput processing. Furthermore, the influence of the filter lobe angle and channel width on the filtration efficiency and outlet flow rate ratio are explored, and an optimisation plan is prepared.
Asunto(s)
Filtración , Filtración/instrumentación , Animales , Saccharomyces cerevisiae/citología , Técnicas Analíticas Microfluídicas/instrumentación , Diseño de Equipo , Dispositivos Laboratorio en un Chip , Tamaño de la PartículaRESUMEN
Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1ATR DNA damage response (DDR). We study DDR and NER in aging cells and find that old cells struggle to repair DNA and activate Mec1ATR. We employ pharmacological and genetic approaches to rescue DDR and NER during aging. Conditions activating Snf1AMPK rescue DDR functionality, but not NER, while inhibition of the TORC1-Sch9S6K axis restores NER and enhances DDR by tuning PP2A activity, specifically in aging cells. Age-related repair deficiency depends on Snf1AMPK-mediated phosphorylation of Sch9S6K on Ser160 and Ser163. PP2A activity in old cells is detrimental for DDR and influences NER by modulating Snf1AMPK and Sch9S6K. Hence, the DDR and repair pathways in aging cells are influenced by the metabolic tuning of opposing AMPK and TORC1 networks and by PP2A activity. Specific Sch9S6K phospho-isoforms control DDR and NER efficiency, specifically during aging.
Asunto(s)
Senescencia Celular , Reparación del ADN , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Daño del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Unicellular organisms such as yeast can survive in very different environments, thanks to a polysaccharide wall that reinforces their extracellular membrane. This wall is not a static structure, as it is expected to be dynamically remodeled according to growth stage, division cycle, environmental osmotic pressure and ageing. It is therefore of great interest to study the mechanics of these organisms, but they are more difficult to study than other mammalian cells, in particular because of their small size (radius of a few microns) and their lack of an adhesion machinery. Using flat cantilevers, we perform compression experiments on single yeast cells (S. cerevisiae) on poly-L-lysine-coated grooved glass plates, in the limit of small deformation using an atomic force microscope (AFM). Thanks to a careful decomposition of force-displacement curves, we extract local scaling exponents that highlight the non-stationary characteristic of the yeast behavior upon compression. Our multi-scale nonlinear analysis of the AFM force-displacement curves provides evidence for non-stationary scaling laws. We propose to model these phenomena based on a two-component elastic system, where each layer follows a different scaling law..
Asunto(s)
Elasticidad , Microscopía de Fuerza Atómica , Modelos Biológicos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Polilisina/química , Fuerza CompresivaRESUMEN
Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred.
Asunto(s)
Mitosis , Saccharomyces cerevisiae , Transducción de Señal , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Nutrientes/metabolismo , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismo , Saccharomycetales/crecimiento & desarrolloRESUMEN
Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in double-membrane vesicles termed autophagosomes, which form from precursors referred to as phagophores. Phagophores grow by lipid influx from the endoplasmic reticulum into Atg9-positive compartments and local lipid synthesis provides lipids for their expansion. How phagophore nucleation and expansion are coordinated with lipid synthesis is unclear. Here, we show that Faa1, an enzyme activating fatty acids, is recruited to Atg9 vesicles by directly binding to negatively charged membranes with a preference for phosphoinositides such as PI3P and PI4P. We define the membrane-binding surface of Faa1 and show that its direct interaction with the membrane is required for its recruitment to phagophores. Furthermore, the physiological localization of Faa1 is key for its efficient catalysis and promotes phagophore expansion. Our results suggest a positive feedback loop coupling phagophore nucleation and expansion to lipid synthesis.
Asunto(s)
Autofagosomas , Ácidos Grasos , Macroautofagia , Autofagia , Ácidos Grasos/metabolismo , Retroalimentación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismoRESUMEN
SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.
Asunto(s)
Fusión de Membrana , Proteínas Munc18 , Proteínas SNARE , Proteínas de Saccharomyces cerevisiae , Proteínas Munc18/metabolismo , Unión Proteica , Proteínas Qa-SNARE/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.
Asunto(s)
Vesículas Citoplasmáticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Munc18/análisis , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismoRESUMEN
Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.
Asunto(s)
Endosomas , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab , Proteínas Activadoras de ras GTPasa , Transporte Biológico , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Vacuolas , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Recent advances in computing power triggered the use of artificial intelligence in image analysis in life sciences. To train these algorithms, a large enough set of certified labeled data is required. The trained neural network is then capable of producing accurate instance segmentation results that will then need to be re-assembled into the original dataset: the entire process requires substantial expertise and time to achieve quantifiable results. To speed-up the process, from cell organelle detection to quantification across electron microscopy modalities, we propose a deep-learning based approach for fast automatic outline segmentation (FAMOUS), that involves organelle detection combined with image morphology, and 3D meshing to automatically segment, visualize and quantify cell organelles within volume electron microscopy datasets. From start to finish, FAMOUS provides full segmentation results within a week on previously unseen datasets. FAMOUS was showcased on a HeLa cell dataset acquired using a focused ion beam scanning electron microscope, and on yeast cells acquired by transmission electron tomography. RESEARCH HIGHLIGHTS: Introducing a rapid, multimodal machine-learning workflow for the automatic segmentation of 3D cell organelles. Successfully applied to a variety of volume electron microscopy datasets and cell lines. Outperforming manual segmentation methods in time and accuracy. Enabling high-throughput quantitative cell biology.
Asunto(s)
Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador , Orgánulos , Orgánulos/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Células HeLa , Microscopía Electrónica/métodos , Imagenología Tridimensional/métodos , Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/citología , Redes Neurales de la Computación , Algoritmos , Microscopía Electrónica de VolumenRESUMEN
The septin cytoskeleton is extensively regulated by posttranslational modifications, such as phosphorylation, to achieve the diversity of architectures including rings, hourglasses, and gauzes. While many of the phosphorylation events of septins have been extensively studied in the budding yeast Saccharomyces cerevisiae, the regulation of the kinases involved remains poorly understood. Here, we show that two septin-associated kinases, the LKB1/PAR-4-related kinase Elm1 and the Nim1/PAR-1-related kinase Gin4, regulate each other at two discrete points of the cell cycle. During bud emergence, Gin4 targets Elm1 to the bud neck via direct binding and phosphorylation to control septin hourglass assembly and stability. During mitosis, Elm1 maintains Gin4 localization via direct binding and phosphorylation to enable timely remodeling of the septin hourglass into a double ring. This mutual control between Gin4 and Elm1 ensures that septin architecture is assembled and remodeled in a temporally controlled manner to perform distinct functions during the cell cycle.
Asunto(s)
Citoesqueleto , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Septinas , Ciclo Celular , Mitosis , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Septinas/genéticaRESUMEN
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of â¼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucariotas/metabolismo , Redes Neurales de la Computación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.
Asunto(s)
Cromatina , Replicación del ADN , Epistasis Genética , Histonas , Saccharomyces cerevisiae , Sitios de Unión , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestructura , Microscopía por Crioelectrón , Replicación del ADN/genética , ADN de Hongos/biosíntesis , ADN de Hongos/química , ADN de Hongos/metabolismo , ADN de Hongos/ultraestructura , Epistasis Genética/genética , Histonas/química , Histonas/metabolismo , Histonas/ultraestructura , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/ultraestructura , Nucleosomas/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.
Asunto(s)
Cromatina , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Meiosis/genética , Mutación , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Lateral diffusion barriers compartmentalize membranes to generate polarity or asymmetrically partition membrane-associated macromolecules. Budding yeasts assemble such barriers in the endoplasmic reticulum (ER) and the outer nuclear envelope at the bud neck to retain aging factors in the mother cell and generate naïve and rejuvenated daughter cells. However, little is known about whether other organelles are similarly compartmentalized. Here, we show that the membranes of mitochondria are laterally compartmentalized at the bud neck and near the cell poles. The barriers in the inner mitochondrial membrane are constitutive, whereas those in the outer membrane form in response to stresses. The strength of mitochondrial diffusion barriers is regulated positively by spatial cues from the septin axis and negatively by retrograde (RTG) signaling. These data indicate that mitochondria are compartmentalized in a fission-independent manner. We propose that these diffusion barriers promote mitochondrial polarity and contribute to mitochondrial quality control.