RESUMO
How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in G1, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. Our quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.
RESUMO
Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes1-4. There are at least two NPC assembly pathways-one during the exit from mitosis and one during nuclear growth in interphase-but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Humanos , Interfase , Mitose , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Espectrometria de FluorescênciaRESUMO
In eukaryotic cells that undergo open mitosis, nuclear pore complex assembly proceeds via two distinct pathways: postmitotic and interphase assembly. Studying both assembly processes is challenging because postmitotic assembly is fast, interphase assembly is rare and sporadic, and assembly intermediates in both pathways are very small with a diameter below 100 nm. Here, we present a protocol for studying nuclear pore complex biogenesis in situ in cultured human cells in a spatiotemporally resolved and quantitative manner by combining live imaging with three-dimensional electron microscopy. The method described here can also be applied for studying other cell cycle-associated events with high spatiotemporal resolution.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Humanos , Interfase , Microscopia Eletrônica , Mitose , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.
Assuntos
Centrossomo/fisiologia , Fuso Acromático/fisiologia , Zigoto/fisiologia , Animais , Bovinos , Embrião de Mamíferos/fisiologia , Genoma/fisiologia , Masculino , Centro Organizador dos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Transdução de Sinais/fisiologia , Espermatozoides/fisiologiaRESUMO
Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of â¼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.
Assuntos
Microscopia de Fluorescência/métodos , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Poro Nuclear/metabolismo , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Humanos , CoesinasRESUMO
Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, the timing of interactions and changes in cellular structure are all crucial to ensure the correct assembly, function and regulation of protein complexes1-4. Imaging of live cells can reveal protein distributions and dynamics but experimental and theoretical challenges have prevented the collection of quantitative data, which are necessary for the formulation of a model of mitosis that comprehensively integrates information and enables the analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries. Here we generate a canonical model of the morphological changes during the mitotic progression of human cells on the basis of four-dimensional image data. We use this model to integrate dynamic three-dimensional concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here to generate a dynamic protein atlas of human cell division is generic; it can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types, and can be conceptually transferred to other cellular functions.
Assuntos
Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Mitose , Edição de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional , Microscopia de Fluorescência , Imagem Molecular , Fatores de TempoRESUMO
At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.
Assuntos
Segregação de Cromossomos , Embrião de Mamíferos/embriologia , Herança Materna/genética , Herança Paterna/genética , Polos do Fuso/metabolismo , Zigoto/metabolismo , Anáfase , Animais , Blastômeros/citologia , Núcleo Celular/metabolismo , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismoRESUMO
The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis. In contrast, fluorescence correlation spectroscopy (FCS) provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius, and oligomerization but lacks the capability for high-throughput application in 4D spatial and temporal imaging. Here we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. These data are processed to yield a calibration curve relating the fluorescence intensities (FIs) of image voxels to the absolute protein abundance. The calibration curve allows the conversion of the arbitrary FIs to protein amounts for all voxels of 4D imaging stacks. Using our workflow, users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared with other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 d.
Assuntos
Células/química , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Automação Laboratorial/métodos , Edição de Genes/métodos , Análise Espaço-TemporalRESUMO
The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing, and dynamic localization during mitosis by fluorescence correlation spectroscopy (FSC)-calibrated live-cell imaging and superresolution microscopy. Although â¼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, â¼195,000 Condensin I complexes dynamically bind in two steps: prometaphase and early anaphase. The two Condensins rarely colocalize at the chromatid axis, where Condensin II is centrally confined, but Condensin I reaches â¼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of â¼450 kb at the chromatid axis, whose size is then reduced by Condensin I binding to â¼90 kb in prometaphase and â¼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.
Assuntos
Adenosina Trifosfatases/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Complexos Multiproteicos/genética , Anáfase/genética , Cromátides/genética , Edição de Genes , HumanosRESUMO
In the version of this Letter originally published, the authors omitted a citation of an early study demonstrating topoisomerase-II-dependent sister chromatid resolution. This reference has now been added to the reference list as reference number 28, and the relevant text has been amended as follows to include its citation: 'Resolution must reflect the removal of sister-sister contacts, and we show here that Topo-IIα-mediated release of DNA catenation plays a major role (Fig. 4), in agreement with previous findings28, whereas, surprisingly, cohesin dissociation is not strictly required (Fig. 3).' Subsequent references have been renumbered. All online versions of the Letter have been updated to reflect this change.
RESUMO
The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.
Assuntos
Núcleo Celular/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/metabolismo , Animais , Membrana Celular/metabolismo , Cromossomos , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/metabolismo , Edição de Genes , Células HeLa , Humanos , Interfase , Cinética , Microscopia Eletrônica de Varredura , Mitose , XenopusRESUMO
Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Genoma Humano/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , CoesinasRESUMO
The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Membrana Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/genética , Animais , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Células HeLa , Humanos , Microscopia de Fluorescência , Membrana Nuclear/química , Membrana Nuclear/ultraestrutura , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestruturaRESUMO
The formation of mitotic chromosomes requires both compaction of chromatin and the resolution of replicated sister chromatids. Compaction occurs during mitotic prophase and prometaphase, and in prophase relies on the activity of condensin II complexes. Exactly when and how sister chromatid resolution occurs has been largely unknown, as has its molecular requirements. Here, we established a method to visualize sister resolution by sequential replication labelling with two distinct nucleotide derivatives. Quantitative three-dimensional imaging then allowed us to measure the resolution of sister chromatids throughout mitosis by calculating their non-overlapping volume within the whole chromosome. Unexpectedly, we found that sister chromatid resolution starts already at the beginning of prophase, proceeds concomitantly with chromatin compaction and is largely completed by the end of prophase. Sister chromatid resolution was abolished by inhibition of topoisomerase IIα and by depleting or preventing mitotic activation of condensin II, whereas blocking cohesin dissociation from chromosomes had little effect. Mitotic sister chromatid resolution is thus an intrinsic part of mitotic chromosome formation in prophase that relies largely on DNA decatenation and shares the molecular requirement for condensin II with prophase compaction.
Assuntos
Cromátides/metabolismo , Mitose/fisiologia , Prometáfase/fisiologia , Prófase/fisiologia , Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Linhagem Celular , Replicação do DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Imageamento Tridimensional/métodos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2ß as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Imagem Molecular , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Membrana Nuclear/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptor de Lamina BRESUMO
The advent of genome-wide RNA interference (RNAi)-based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome on the most likely candidates. Although different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyzes these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function-mitotic chromosome condensation-and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes, including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are powerful tools to integrate public biological data and predict genes involved in cellular functions of interest.
Assuntos
Segregação de Cromossomos/genética , Cromossomos/genética , Biologia Computacional/métodos , Genoma , Células HeLa , Humanos , Microscopia Confocal , Mitose , Fenótipo , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/genética , SoftwareRESUMO
The aim of this paper is to detail the development of a novel tracking framework that is able to extract the cell motility indicators and to determine the cellular division (mitosis) events in large time-lapse phase-contrast image sequences. To address the challenges induced by nonstructured (random) motion, cellular agglomeration, and cellular mitosis, the process of automatic (unsupervised) cell tracking is carried out in a sequential manner, where the interframe cell association is achieved by assessing the variation in the local cellular structures in consecutive frames of the image sequence. In our study, a strong emphasis has been placed on the robust use of the topological information in the cellular tracking process and in the development of targeted pattern recognition techniques that were designed to redress the problems caused by segmentation errors, and to precisely identify mitosis using a backward (reversed) tracking strategy. The proposed algorithm has been evaluated on dense phase-contrast cellular data and the experimental results indicate that the proposed algorithm is able to accurately track epithelial and endothelial cells in time-lapse image sequences that are characterized by low contrast and high level of noise. Our algorithm achieved 86.10% overall tracking accuracy and 90.12% mitosis detection accuracy.
Assuntos
Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Mitose/fisiologia , Imagem com Lapso de Tempo/métodos , Algoritmos , Animais , Cães , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Madin Darby de Rim CaninoRESUMO
Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.
Assuntos
Tecido Conjuntivo/embriologia , Coturnix/embriologia , Endocárdio/embriologia , Organogênese , Animais , Fibrilinas , Fibronectinas/metabolismo , Mesoderma/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Morfogênese , Receptor de TIE-1/metabolismoRESUMO
The process required to track cellular structures is a key task in the study of cell migration. This allows the accurate estimation of motility indicators that help in the understanding of mechanisms behind various biological processes. This paper reports a particle-based fully automatic tracking framework that is able to quantify the motility of living cells in time-lapse images. Contrary to the standard tracking methods based on predefined motion models, in this paper we reformulate the tracking mechanism as a data driven optimization process to remove its reliance on a priory motion models. The proposed method has been evaluated using 2D and 3D deconvolved epifluorescent in-vivo image sequences that describe the development of the quail embryo.