RESUMEN
Chromatin is organized in the nucleus via CTCF loops and compartmental domains. Here, we compare different cell types to identify distinct paradigms of compartmental domain formation in human tissues. We identify and quantify compartmental forces correlated with histone modifications characteristic of transcriptional activity and previously underappreciated roles for distinct compartmental domains correlated with the presence of H3K27me3 and H3K9me3, respectively. We present a computer simulation model capable of predicting compartmental organization based on the biochemical characteristics of independent chromatin features. Using this model, we show that the underlying forces responsible for compartmental domain formation in human cells are conserved and that the diverse compartmentalization patterns seen across cell types are due to differences in chromatin features. We extend these findings to Drosophila to suggest that the same principles are at work beyond humans. These results offer mechanistic insights into the fundamental forces driving the 3D organization of the genome.
Asunto(s)
Compartimento Celular/genética , Genoma Humano , Imagenología Tridimensional , Animales , Cromatina/metabolismo , Cromosomas Humanos Par 14/genética , Drosophila/genética , Genoma de los Insectos , Células HCT116 , Código de Histonas/genética , Humanos , Transcripción GenéticaRESUMEN
Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Proteínas Portadoras/genética , Compartimento Celular/genética , Espacio Extracelular/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Microfluídica , Morfogénesis/genética , Transducción de Señal/genética , Transcitosis/genéticaRESUMEN
Bacterial microcompartments are organelle-like structures composed entirely of proteins. They have evolved to carry out several distinct and specialized metabolic functions in a wide variety of bacteria. Their outer shell is constructed from thousands of tessellating protein subunits, encapsulating enzymes that carry out the internal metabolic reactions. The shell proteins are varied, with single, tandem and permuted versions of the PF00936 protein family domain comprising the primary structural component of their polyhedral architecture, which is reminiscent of a viral capsid. While considerable amounts of structural and biophysical data have been generated in the last 15 years, the existing functionalities of current resources have limited our ability to rapidly understand the functional and structural properties of microcompartments (MCPs) and their diversity. In order to make the remarkable structural features of bacterial microcompartments accessible to a broad community of scientists and non-specialists, we developed MCPdb: The Bacterial Microcompartment Database (https://mcpdb.mbi.ucla.edu/). MCPdb is a comprehensive resource that categorizes and organizes known microcompartment protein structures and their larger assemblies. To emphasize the critical roles symmetric assembly and architecture play in microcompartment function, each structure in the MCPdb is validated and annotated with respect to: (1) its predicted natural assembly state (2) tertiary structure and topology and (3) the metabolic compartment type from which it derives. The current database includes 163 structures and is available to the public with the anticipation that it will serve as a growing resource for scientists interested in understanding protein-based metabolic organelles in bacteria.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/genética , Compartimento Celular/genética , Bases de Datos Genéticas , Secuencia de Aminoácidos/genética , Bacterias/clasificación , Proteínas Bacterianas/clasificación , Orgánulos/genética , Dominios Proteicos/genéticaRESUMEN
Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies1-4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.
Asunto(s)
Compartimento Celular/genética , Núcleo Celular/genética , Genómica/métodos , Células Madre Embrionarias de Ratones/citología , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Cromosomas de los Mamíferos/genética , Células Clonales/citología , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos , Histonas/metabolismo , Lisina/metabolismo , Masculino , Ratones , Factores de TiempoRESUMEN
Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.
Asunto(s)
Gránulos Citoplasmáticos/genética , Polirribosomas/genética , Ribonucleoproteínas/genética , Estrés Fisiológico/genética , Compartimento Celular/genética , Membrana Celular/genética , Citoplasma/genética , Humanos , Microextracción en Fase Líquida , ARN Mensajero/genéticaRESUMEN
Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2CTD is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.
Asunto(s)
Compartimento Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/citología , Drosophila/genética , ARN/metabolismo , Factores de Transcripción/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismo , Animales , Compartimento Celular/genética , Línea Celular , Proteínas de Unión al ADN/química , Drosophila/metabolismo , Proteínas de Drosophila/química , Femenino , Humanos , Masculino , Ratones , Conformación de Ácido Nucleico , ARN/genética , Factores de Transcripción/químicaRESUMEN
Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein-peptide pair. Escherichia coli cells co-expressing protein-peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein-peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher-SpyTag pair and co-selecting for functionally interacting variants.
Asunto(s)
Evolución Molecular Dirigida , Escherichia coli/genética , Péptidos/genética , Mapas de Interacción de Proteínas/genética , Compartimento Celular/genética , Replicación del ADN/genética , Regulación Bacteriana de la Expresión Génica/genética , Genotipo , Luciferasas/genética , Fenotipo , Ingeniería de Proteínas , Polimerasa Taq/genéticaRESUMEN
SOX9 plays an important role in chondrocyte differentiation and, in the developing axial skeleton, maintains the notochord and the demarcation of intervertebral disc compartments. Diminished expression is linked to campomelic dysplasia, resulting in severe scoliosis and progressive disc degeneration. However, the specific functions of SOX9 in the adult spinal column and disc are largely unknown. Accordingly, employing a strategy to conditionally delete Sox9 in Acan-expressing cells (AcanCreERT2Sox9fl/fl), we delineated these functions in the adult intervertebral disc. AcanCreERT2Sox9fl/fl mice (Sox9cKO) showed extensive and progressive remodeling of the extracellular matrix in nucleus pulposus (NP) and annulus fibrosus (AF), consistent with human disc degeneration. Progressive degeneration of the cartilaginous endplates (EP) was also evident in Sox9cKO mice, and it preceded morphological changes seen in the NP and AF compartments. Fate mapping using tdTomato reporter, EdU chase, and quantitative immunohistological studies demonstrated that SOX9 is crucial for disc cell survival and phenotype maintenance. Microarray analysis showed that Sox9 regulated distinct compartment-specific transcriptomic landscapes, with prominent contributions to the ECM, cytoskeleton-related, and metabolic pathways in the NP and ion transport, the cell cycle, and signaling pathways in the AF. In summary, our work provides new insights into disc degeneration in Sox9cKO mice at the cellular, molecular, and transcriptional levels, underscoring tissue-specific roles of this transcription factor. Our findings may direct future cell therapies targeting SOX9 to mitigate disc degeneration.
Asunto(s)
Matriz Ósea/metabolismo , Degeneración del Disco Intervertebral/genética , Factor de Transcripción SOX9/genética , Transcriptoma/genética , Animales , Apoptosis/genética , Matriz Ósea/patología , Compartimento Celular/genética , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrocitos/patología , Matriz Extracelular/genética , Regulación de la Expresión Génica/genética , Humanos , Degeneración del Disco Intervertebral/patología , Ratones , Ratones NoqueadosRESUMEN
Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity.IMPORTANCEHelicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.
Asunto(s)
Compartimento Celular/genética , Endorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasas/genética , Compartimento Celular/fisiología , Helicobacter pylori/patogenicidad , Estabilidad del ARN , ARN Bacteriano/genética , ARN Mensajero , Ribonucleasas/metabolismoRESUMEN
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
Asunto(s)
Carcinogénesis/metabolismo , Carcinoma Hepatocelular/genética , Compartimento Celular/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas del Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Transducción de Señal , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinoma Hepatocelular/metabolismo , Compartimento Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , AMP Cíclico/farmacología , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Oncogenes/genética , Dominios Proteicos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión , Espectroscopía Infrarroja por Transformada de Fourier , Imagen de Lapso de Tiempo/métodosRESUMEN
Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.
Asunto(s)
Ciclo Celular/genética , Polaridad Celular/genética , Eremothecium/metabolismo , Proteínas Fúngicas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Alelos , Secuencia de Bases , Compartimento Celular/genética , Citosol/metabolismo , Citosol/ultraestructura , Eremothecium/genética , Eremothecium/ultraestructura , Proteínas Fúngicas/genética , Expresión Génica , Calor , Mutación , Fosforilación , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Estrés Fisiológico/genéticaRESUMEN
Cells compartmentalize their genome into active and inactive parts that contain distinct histone marks and are differently packaged. Recent work has shed new light on the mechanisms that drive this type of chromatin patterning and the properties of the resulting domains. Biophysical concepts such as liquid-liquid phase separation, polymer looping and collapse, molecular crowding and viscoelasticity are increasingly used to describe experimental observations. Accordingly, it is becoming clear that the physicochemical properties of the nuclear interior are relevant for understanding chromatin compartmentalization. Here, I discuss recent insights into the properties of chromatin subcompartments obtained with complementary techniques on different scales, and relate them to models for functional chromatin patterning.
Asunto(s)
Fenómenos Biofísicos , Compartimento Celular/genética , Núcleo Celular/genética , Cromatina/genética , Animales , Genoma/genética , HumanosRESUMEN
TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liquid-liquid phase separation to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signalling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase separation of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-separation mechanism for efficient and specific transcriptional activation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Quinasa 9 Dependiente de la Ciclina/genética , Proteínas de Unión al ADN/genética , Subunidad 1 del Complejo Mediador/genética , Proteínas Musculares/genética , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Musculares/metabolismo , Fosforilación , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factores de Transcripción de Dominio TEA , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF-CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Telofase , Adenosina Trifosfatasas/metabolismo , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Citocinesis/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Células HeLa , Humanos , Interfase , Complejos Multiproteicos/metabolismo , Fase S , CohesinasRESUMEN
Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.
Asunto(s)
Proteínas de Arabidopsis/genética , Calmodulina/genética , Compartimento Celular/genética , Proteínas de Cloroplastos/genética , Proteínas de la Membrana/genética , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Sitios de Unión/genética , Señalización del Calcio/genética , Calmodulina/química , Proteínas de Cloroplastos/química , Cloroplastos/química , Cloroplastos/genética , Citosol/química , Proteínas de la Membrana/química , Plastidios/química , Plastidios/genética , Unión Proteica/genéticaRESUMEN
CTP synthase (CTPS) has been demonstrated to form evolutionarily-conserved filamentous structures termed cytoophidia whose exact cellular functions remain unclear, but they may play a role in intracellular compartmentalization. We have previously shown that the mammalian target of rapamycin complex 1 (mTORC1)-S6K1 pathway mediates cytoophidium assembly in mammalian cells. Here, using the fission yeast Schizosaccharomyces pombe as a model of a unicellular eukaryote, we demonstrate that the target of rapamycin (TOR)-signaling pathway regulates cytoophidium formation (from the S. pombe CTPS ortholog Cts1) also in S. pombe Conducting a systematic analysis of all viable single TOR subunit-knockout mutants and of several major downstream effector proteins, we found that Cts1 cytoophidia are significantly shortened and often dissociate when TOR is defective. We also found that the activities of the downstream effector kinases of the TORC1 pathway, Sck1, Sck2, and Psk1 S6, as well as of the S6K/AGC kinase Gad8, the major downstream effector kinase of the TORC2 pathway, are necessary for proper cytoophidium filament formation. Interestingly, we observed that the Crf1 transcriptional corepressor for ribosomal genes is a strong effector of Cts1 filamentation. Our findings connect TOR signaling, a major pathway required for cell growth, with the compartmentalization of the essential nucleotide synthesis enzyme CTPS, and we uncover differences in the regulation of its filamentation among higher multicellular and unicellular eukaryotic systems.
Asunto(s)
Ligasas de Carbono-Nitrógeno/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Schizosaccharomyces/genética , Ligasas de Carbono-Nitrógeno/química , Compartimento Celular/genética , Citoplasma/genética , Técnicas de Inactivación de Genes , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Receptores de Hormona Liberadora de Corticotropina/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina2,4, preferential attraction of similar chromatin to each other1,4-12, higher levels of chromatin mobility in active chromatin13-15 and transcription-related clustering of euchromatin16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases.
Asunto(s)
Compartimento Celular , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Animales , Compartimento Celular/genética , Núcleo Celular/genética , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , Ratones , Modelos Biológicos , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Factores de TiempoRESUMEN
Genome expression and stability are dependent on biological processes that control repetitive DNA sequences and nuclear compartmentalization. The phase separation of macromolecules has recently emerged as a major player in the control of biological pathways. Here, we summarize recent studies that collectively reveal intersections between phase separation, repetitive DNA elements, and nuclear compartments. These intersections modulate fundamental processes, including gene expression, DNA repair, and cellular lifespan, in the context of health and diseases such as cancer and neurodegeneration.
Asunto(s)
Regulación de la Expresión Génica/genética , Genoma/genética , Neoplasias/genética , Enfermedades Neurodegenerativas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Envejecimiento/genética , Compartimento Celular/genética , Reparación del ADN/genética , Sitios Genéticos/genética , HumanosRESUMEN
Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.
Asunto(s)
Antiportadores/genética , Macrófagos/metabolismo , Proteínas de Transporte de Membrana/genética , Microglía/metabolismo , Fagosomas/ultraestructura , Animales , Apoptosis/genética , Compartimento Celular/genética , Células HeLa , Humanos , Macrófagos/ultraestructura , Ratones , Microglía/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Fagocitos/ultraestructura , Fagocitosis/genética , Fagosomas/genética , Células RAW 264.7 , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.