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The individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase in human cells. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces, driven by co-condensation of Ki-67 and RNA, promotes clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.
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Dynamic processes involving biomolecules are essential for the function of the cell. Here, we introduce an integrative method for computing models of these processes based on multiple heterogeneous sources of information, including time-resolved experimental data and physical models of dynamic processes. We first compute integrative structure models at fixed time points and then optimally select and connect these snapshots into a series of trajectories that optimize the likelihood of both the snapshots and transitions between them. The method is demonstrated by application to the assembly process of the human Nuclear Pore Complex in the context of the reforming nuclear envelope during mitotic cell division, based on live-cell correlated electron tomography, bulk fluorescence correlation spectroscopy-calibrated quantitative live imaging, and a structural model of the fully-assembled Nuclear Pore Complex. Modeling of the assembly process improves the model precision over static integrative structure modeling alone. The method is applicable to a wide range of time-dependent systems in cell biology, and is available to the broader scientific community through an implementation in the open source Integrative Modeling Platform software.
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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.
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In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.
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Pesquisadores , Humanos , Mobilidade Ocupacional , Pesquisa Biomédica/métodos , Escolha da ProfissãoRESUMO
Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.
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Redes Neurais de Computação , Organelas , Humanos , Microscopia Crioeletrônica/métodos , Ligantes , Organelas/ultraestrutura , Tomografia com Microscopia Eletrônica/métodosRESUMO
Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.
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Caenorhabditis elegans , Microscopia , Animais , Camundongos , Peixe-Zebra , Luz , LasersRESUMO
MOTIVATION: The nuclear pore complex (NPC) is the only passageway for macromolecules between nucleus and cytoplasm, and an important reference standard in microscopy: it is massive and stereotypically arranged. The average architecture of NPC proteins has been resolved with pseudoatomic precision, however observed NPC heterogeneities evidence a high degree of divergence from this average. Single-molecule localization microscopy (SMLM) images NPCs at protein-level resolution, whereupon image analysis software studies NPC variability. However, the true picture of this variability is unknown. In quantitative image analysis experiments, it is thus difficult to distinguish intrinsically high SMLM noise from variability of the underlying structure. RESULTS: We introduce CIR4MICS ('ceramics', Configurable, Irregular Rings FOR MICroscopy Simulations), a pipeline that synthesizes ground truth datasets of structurally variable NPCs based on architectural models of the true NPC. Users can select one or more N- or C-terminally tagged NPC proteins, and simulate a wide range of geometric variations. We also represent the NPC as a spring-model such that arbitrary deforming forces, of user-defined magnitudes, simulate irregularly shaped variations. Further, we provide annotated reference datasets of simulated human NPCs, which facilitate a side-by-side comparison with real data. To demonstrate, we synthetically replicate a geometric analysis of real NPC radii and reveal that a range of simulated variability parameters can lead to observed results. Our simulator is therefore valuable to test the capabilities of image analysis methods, as well as to inform experimentalists about the requirements of hypothesis-driven imaging studies. AVAILABILITY AND IMPLEMENTATION: Code: https://github.com/uhlmanngroup/cir4mics. Simulated data: BioStudies S-BSST1058.
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Microscopia , Poro Nuclear , Humanos , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Imagem Individual de Molécula/métodos , SoftwareRESUMO
Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.
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Blastocisto , Implantação do Embrião , Feminino , Camundongos , Animais , Trofoblastos , Útero , Desenvolvimento Embrionário , MamíferosRESUMO
Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.
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Desenvolvimento Embrionário , Microscopia , Animais , Camundongos , Microscopia/métodos , Drosophila , Embrião não Mamífero , Imageamento Tridimensional/métodosRESUMO
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.
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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
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.
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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.
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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/metabolismoAssuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Metadados , Algoritmos , Animais , Congressos como Assunto , Microscopia Crioeletrônica/métodos , Mineração de Dados/métodos , Bases de Dados Factuais , Diagnóstico por Imagem/métodos , Humanos , Microscopia , SoftwareRESUMO
Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single-molecule sensitivity. Here, we introduce Proximity-PAINT (pPAINT), a variation of the super-resolution microscopy technique DNA-PAINT. pPAINT uses a split-docking-site configuration to detect spatial proximity with high sensitivity, low false-positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha- and beta-tubulin in microtubules using super-resolution detection.
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Microscopia de Fluorescência/métodos , Tubulina (Proteína)/análise , Anticorpos/imunologia , DNA/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microtúbulos/química , Nanoestruturas/química , Hibridização de Ácido Nucleico , Tubulina (Proteína)/imunologiaRESUMO
Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
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Terapia Baseada em Transplante de Células e Tecidos , Atenção à Saúde/métodos , Atenção à Saúde/tendências , Medicina/métodos , Medicina/tendências , Patologia , Análise de Célula Única , Inteligência Artificial , Atenção à Saúde/ética , Atenção à Saúde/normas , Diagnóstico Precoce , Educação Médica , Europa (Continente) , Feminino , Saúde , Humanos , Legislação Médica , Masculino , Medicina/normasRESUMO
We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.