RESUMEN
Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.
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Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.
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The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Transportador de Aminoácidos Neutros Grandes 1 , Animales , Ratones , Diferenciación Celular/genética , Proliferación Celular/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.
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Bacillus subtilis , Proteínas Bacterianas , Bacillus subtilis/metabolismo , Activación Transcripcional , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , División Celular/genética , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.
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Oído Interno , Canales de Potasio de Rectificación Interna , Animales , Ratones , Estría Vascular/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Cóclea/metabolismo , Oído Interno/metabolismo , Ratones Transgénicos , Mamíferos/metabolismoRESUMEN
Fluorescence microscopy is an invaluable tool in biology, yet its performance is compromised when the wavefront of light is distorted due to optical imperfections or the refractile nature of the sample. Such optical aberrations can dramatically lower the information content of images by degrading image contrast, resolution, and signal. Adaptive optics (AO) methods can sense and subsequently cancel the aberrated wavefront, but are too complex, inefficient, slow, or expensive for routine adoption by most labs. Here we introduce a rapid, sensitive, and robust wavefront sensing scheme based on phase diversity, a method successfully deployed in astronomy but underused in microscopy. Our method enables accurate wavefront sensing to less than λ/35 root mean square (RMS) error with few measurements, and AO with no additional hardware besides a corrective element. After validating the method with simulations, we demonstrate calibration of a deformable mirror > 100-fold faster than comparable methods (corresponding to wavefront sensing on the ~100 ms scale), and sensing and subsequent correction of severe aberrations (RMS wavefront distortion exceeding λ/2), restoring diffraction-limited imaging on extended biological samples.
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During wound healing and surgical implantation, the body establishes a delicate balance between immune activation to fight off infection and clear debris and immune tolerance to control reactivity against self-tissue. Nonetheless, how such a balance is achieved is not well understood. Here we describe that pro-regenerative biomaterials for muscle injury treatment promote the proliferation of a BATF3-dependent CD103+XCR1+CD206+CD301b+ dendritic cell population associated with cross-presentation and self-tolerance. Upregulation of E-cadherin, the ligand for CD103, and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Muscle injury recruited natural killer cells that produced Xcl1 when stimulated with fragmented extracellular matrix. Without cross-presenting cells, T-cell activation increases, pro-regenerative macrophage polarization decreases and there are alterations in myogenesis, adipogenesis, fibrosis and increased muscle calcification. These results, previously observed in cancer progression, suggest a fundamental mechanism of immune regulation in trauma and material implantation with implications for both short- and long-term injury recovery.
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Materiales Biocompatibles , Células Dendríticas , Materiales Biocompatibles/farmacologíaRESUMEN
Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics into the imaging path. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations to show that applying the trained 'de-aberration' networks outperforms alternative methods, and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos.
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The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.
RESUMEN
When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that a unique feature of the sporulation septum, defined by the cell division machinery, drives the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.
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Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.
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Neoplasias Colorrectales , Inhibidores de Topoisomerasa I , Humanos , Inhibidores de Topoisomerasa I/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ligasas/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al ARNRESUMEN
Tissue clearing of whole intact organs has enhanced imaging by enabling the exploration of tissue structure at a subcellular level in three-dimensional space. Although clearing and imaging of the whole organ have been used to study tissue biology, the microenvironment in which cells evolve to adapt to biomaterial implants or allografts in the body is poorly understood. Obtaining high-resolution information from complex cell-biomaterial interactions with volumetric landscapes represents a key challenge in the fields of biomaterials and regenerative medicine. To provide a new approach to examine how tissue responds to biomaterial implants, we apply cleared tissue light-sheet microscopy and three-dimensional reconstruction to utilize the wealth of autofluorescence information for visualizing and contrasting anatomical structures. This study demonstrates the adaptability of the clearing and imaging technique to provide sub-cellular resolution (0.6 µm isotropic) 3D maps of various tissue types, using samples from fully intact peritoneal organs to volumetric muscle loss injury specimens. Specifically, in the volumetric muscle loss injury model, we provide 3D visualization of the implanted extracellular matrix biomaterial in the wound bed of the quadricep muscle groups and further apply computational-driven image classification to analyze the autofluorescence spectrum at multiple emission wavelengths to categorize tissue types at the injured site interacting with the biomaterial scaffolds.
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Materiales Biocompatibles , Microscopía , Microscopía/métodos , Matriz Extracelular , Aprendizaje Automático , Imagenología Tridimensional/métodosRESUMEN
Patient-derived induced pluripotent stem cells (iPSCs), carrying the genetic information of the disease and capable of differentiating into multilineages in vitro, are valuable for disease modeling. 3D bioprinting enables the assembly of the cell-laden hydrogel into hierarchically three-dimensional architectures that recapitulate the natural tissues and organs. Investigation of iPSC-derived physiological and pathological models constructed by 3D bioprinting is a fast-growing field still in its infancy. Distinctly from cell lines and adult stem cells, iPSCs and iPSC-derived cells are more susceptible to external stimuli which can disturb the differentiation, maturation, and organization of iPSCs and their progeny. Here we discuss the fitness of iPSCs and 3D bioprinting from the perspective of bioinks and printing technologies. We provide a timely review of the progress of 3D bioprinting iPSC-derived physiological and pathological models by exemplifying the relatively prosperous cardiac and neurological fields. We also discuss scientific rigors and highlight the remaining issues to offer a guiding framework for bioprinting-assisted personalized medicine.
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Bioimpresión , Células Madre Pluripotentes Inducidas , Humanos , Ingeniería de Tejidos/métodos , Bioimpresión/métodos , Diferenciación Celular , Línea CelularRESUMEN
The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.
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[This corrects the article DOI: 10.1016/j.bioactmat.2020.08.035.].
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The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to â¼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with â¼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving â¼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.
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Imagenología Tridimensional , Iluminación , Microscopía Fluorescente/métodos , Imagenología Tridimensional/métodos , Citoesqueleto , LisosomasRESUMEN
We present Richardson-Lucy network (RLN), a fast and lightweight deep learning method for three-dimensional fluorescence microscopy deconvolution. RLN combines the traditional Richardson-Lucy iteration with a fully convolutional network structure, establishing a connection to the image formation process and thereby improving network performance. Containing only roughly 16,000 parameters, RLN enables four- to 50-fold faster processing than purely data-driven networks with many more parameters. By visual and quantitative analysis, we show that RLN provides better deconvolution, better generalizability and fewer artifacts than other networks, especially along the axial dimension. RLN outperforms classic Richardson-Lucy deconvolution on volumes contaminated with severe out of focus fluorescence or noise and provides four- to sixfold faster reconstructions of large, cleared-tissue datasets than classic multi-view pipelines. We demonstrate RLN's performance on cells, tissues and embryos imaged with widefield-, light-sheet-, confocal- and super-resolution microscopy.
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Algoritmos , Aprendizaje Profundo , Artefactos , Microscopía Fluorescente , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Accurately obtaining the spatial distribution information of fruit tree planting is of great significance to the development of fruit tree growth monitoring, disease and pest control, and yield estimation. In this study, the Sentenel-2 multispectral remote sensing imageries of different months during the growth period of the fruit trees were used as the data source, and single month vegetation indices, accumulated monthly vegetation indices (∑VIs), and difference vegetation indices between adjacent months (∆VIs) were constructed as input variables. Four conventional vegetation indices of NDVI, PSRI, GNDVI, and RVI and four improved vegetation indices of NDVIre1, NDVIre2, NDVIre3, and NDVIre4 based on the red-edge band were selected to construct a decision tree classification model combined with machine learning technology. Through the analysis of vegetation indices under different treatments and different months, combined with the attribute of Feature_importances_, the vegetation indices of different periods with high contribution were selected as input features, and the Max_depth values of the decision tree model were determined by the hyperparameter learning curve. The results have shown that when the Max_depth value of the decision tree model of the vegetation indices under the three treatments was 6, 8, and 8, the model classification was the best. The accuracy of the three vegetation index processing models on the training set were 0.8936, 0.9153, and 0.8887, and the accuracy on the test set were 0.8355, 0.7611, and 0.7940, respectively. This method could be applied to remote sensing classification of fruit trees in a large area, and could provide effective technical means for monitoring fruit tree planting areas with medium and high resolution remote sensing imageries.
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Frutas , Tecnología de Sensores Remotos , Tecnología de Sensores Remotos/métodosRESUMEN
Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Músculos/metabolismoRESUMEN
Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.