RESUMO
Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.
Assuntos
Inteligência Artificial , Microscopia , Humanos , Microscopia/métodos , Imageamento Tridimensional/métodos , SinapsesRESUMO
The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.
Assuntos
Cromatina/genética , Proteínas de Homeodomínio/genética , Proteínas Intrinsicamente Desordenadas/genética , Neoplasias/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Translocação Genética , Animais , Carcinogênese , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Single-molecule localization microscopy (SMLM) has had remarkable success in imaging cellular structures with nanometer resolution, but standard analysis algorithms require sparse emitters, which limits imaging speed and labeling density. Here, we overcome this major limitation using deep learning. We developed DECODE (deep context dependent), a computational tool that can localize single emitters at high density in three dimensions with highest accuracy for a large range of imaging modalities and conditions. In a public software benchmark competition, it outperformed all other fitters on 12 out of 12 datasets when comparing both detection accuracy and localization error, often by a substantial margin. DECODE allowed us to acquire fast dynamic live-cell SMLM data with reduced light exposure and to image microtubules at ultra-high labeling density. Packaged for simple installation and use, DECODE will enable many laboratories to reduce imaging times and increase localization density in SMLM.
Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Animais , Células COS , Chlorocebus aethiops , Bases de Dados Factuais , SoftwareRESUMO
To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.
Assuntos
DNA/metabolismo , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia/métodos , Coloração Cromossômica , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Processamento de Imagem Assistida por Computador , Análise de Sequência de DNA/métodosRESUMO
The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for ß-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.
Assuntos
Microscopia Confocal , Imagem Molecular/métodos , Organelas/metabolismo , Biologia de Sistemas , Animais , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Cor , Citoesqueleto , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Organelas/química , Peroxissomos/metabolismo , Análise Espaço-TemporalRESUMO
Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 µm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.
Assuntos
Membrana Celular/ultraestrutura , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Mitocôndrias/ultraestrutura , Animais , Células COS , Chlorocebus aethiops , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Células LLC-PK1 , Suínos , Peixe-Zebra/embriologiaRESUMO
Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.
Assuntos
Adesões Focais/fisiologia , Animais , Fenômenos Biofísicos , Fibroblastos/citologia , Fibroblastos/fisiologia , Análise de Elementos Finitos , Proteínas de Fluorescência Verde/metabolismo , Mecanotransdução Celular/fisiologia , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Proteínas Recombinantes/metabolismo , RotaçãoRESUMO
Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Osteogênese/efeitos dos fármacos , ProteóliseRESUMO
Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.
Assuntos
Imageamento Tridimensional , Microscopia de Fluorescência , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Artefatos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Humanos , Animais , Simulação por ComputadorRESUMO
Significance: Lattice light sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing 3D images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct for the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy (SIM) information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show utilizing transfer functions with the same axial offset as that which was present during the data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.
RESUMO
Cell biology emerges from spatiotemporally coordinated molecular processes. Recent advances in live-cell microscopy, fueled by a surge in optical, molecular, and computational technologies, have enabled dynamic observations from single molecules to whole organisms. Despite technological leaps, there is still an untapped opportunity to fully leverage their capabilities toward biological insight. We highlight how single-molecule imaging has transformed our understanding of biological processes, with a focus on chromatin organization and transcription in the nucleus. We describe how this was enabled by the close integration of new imaging techniques with analysis tools and discuss the challenges to make a comparable impact at larger scales from organelles to organisms. By highlighting recent successful examples, we describe an outlook of ever-increasing data and the need for seamless integration between dataset visualization and quantification to realize the full potential warranted by advances in new imaging technologies.
RESUMO
The nucleus coordinates many different processes. Visualizing how these are spatially organized requires imaging protein complexes, epigenetic marks, and DNA across scales from single molecules to the whole nucleus. To accomplish this, we developed a multiplexed imaging protocol to localize 13 different nuclear targets with nanometer precision in single cells. We show that nuclear specification into active and repressive states exists along a spectrum of length scales, emerging below one micron and becoming strengthened at the nanoscale with unique organizational principles in both heterochromatin and euchromatin. HP1-α was positively correlated with DNA at the microscale but uncorrelated at the nanoscale. RNA Polymerase II, p300, and CDK9 were positively correlated at the microscale but became partitioned below 300 nm. Perturbing histone acetylation or transcription disrupted nanoscale organization but had less effect at the microscale. We envision that our imaging and analysis pipeline will be useful to reveal the organizational principles not only of the cell nucleus but also other cellular compartments.
RESUMO
All cells must detect, interpret and adapt to multiple and concurrent stimuli. While signaling pathways are highly specialized, different pathways often share components or have components with overlapping functions. In the yeast Saccharomyces cerevisiae, the high osmolarity glycerol (HOG) pathway has two seemingly redundant branches, mediated by Sln1 and Sho1. Both branches are activated by osmotic pressure, leading to phosphorylation of the MAPKs Hog1 and Kss1. The mating pathway is activated by pheromone, leading to phosphorylation of the MAPKs Fus3 and Kss1. Given that Kss1 is shared by the two pathways, we investigated its role in signal coordination. We activated both pathways with a combination of salt and pheromone, in cells lacking the shared MAPK and in cells lacking either of the redundant branches of the HOG pathway. By systematically evaluating MAPK activation, translocation, and transcription programs, we determined that Sho1 mediates cross talk between the HOG and mating pathways and does so through Kss1. Further, we show that Kss1 initiates a transcriptional program that is distinct from that induced by Hog1 and Fus3. Our findings reveal how redundant and shared components coordinate concurrent signals and thereby adapt to sudden environmental changes.
RESUMO
In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.
Assuntos
Cromatina , Nucleossomos , Imagem Individual de Molécula , Nucleossomos/metabolismo , Cromatina/metabolismo , Cromatina/química , Humanos , Imagem Individual de Molécula/métodos , Núcleo Celular/metabolismo , Histonas/metabolismo , Células HeLa , DifusãoRESUMO
Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.
RESUMO
Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of â¼0.4 mm long, â¼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.
Assuntos
Músculo Liso/citologia , Sistema Respiratório/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Asma/fisiopatologia , Técnicas de Cocultura , Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Contração Muscular/fisiologia , Células NIH 3T3 , Estresse Mecânico , Engenharia Tecidual/métodosRESUMO
Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.