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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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Investigadores , Humanos , Movilidad Laboral , Investigación Biomédica/métodos , Selección de ProfesiónRESUMEN
OBJECTIVE: Ultrasound is being researched as a method to modulate the brain. Studies of the interaction of sound with neurons support the hypothesis that mechanosensitive ion channels play an important role in ultrasound neuromodulation. The response of cells other than neurons (e.g., astrocytes, pericytes and endothelial cells) have not been fully characterized, despite playing an important role in brain function. METHODS: To address this gap in knowledge, we examined cultured murine primary cortical neurons, astrocytes, endothelial cells and pericytes in an in vitro widefield microscopy setup during application of a 500 ms burst of 250 kHz focused ultrasound over a pressure range known to elicit neuromodulation. We examined cell membrane health in response to a range of pulses and used optical calcium indicators in conjunction with pharmacological antagonists to selectively block different groups of thermo- and mechanosensitive ion channels known to be responsive to ultrasound. RESULTS: All cell types experienced an increase in calcium fluorescence in response to ultrasound. Gadolinium (Gad), 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RR) reduced the percentage of responding neurons and magnitude of response. The percentage of astrocytes responding was significantly lowered only by Gad, whereas both 2-APB and Gad decreased the amplitude of the fluorescence response. 2-APB decreased the percentage of responding endothelial cells, whereas only Gad reduced the magnitude of responses. Pericytes exposed to RR or Gad were less likely to respond to stimulation. RR had no detectable effect on the magnitude of the pericyte responses while 2-APB and Gad significantly decreased the fluorescence intensity, despite not affecting the percentage responding. CONCLUSION: Our study highlights the role of non-neuronal cells during FUS neuromodulation. All of the investigated cell types are sensitive to mechanical ultrasound stimulation and rely on mechanosensitive ion channels to undergo ultrasound neuromodulation.
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Calcio , Pericitos , Ratones , Animales , Calcio/metabolismo , Pericitos/metabolismo , Células Endoteliales/metabolismo , Neuronas , Canales Iónicos/metabolismo , Células CultivadasRESUMEN
To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.
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Células Epiteliales , Humanos , Animales , Ratones , Porcinos , Microvellosidades/metabolismo , Células Epiteliales/metabolismoRESUMEN
Differentiated transporting epithelial cells present an extensive apical array of microvilli - a "brush border" - where neighboring microvilli are linked together by intermicrovillar adhesion complexes (IMACs) composed of protocadherins CDHR2 and CDHR5. Although loss-of-function studies provide strong evidence that IMAC function is needed to build a mature brush border, how the IMAC contributes to the stabilization and accumulation of nascent microvilli remains unclear. We found that, early in differentiation, the apical surface exhibits a marginal accumulation of microvilli, characterized by higher packing density relative to medial regions of the surface. While medial microvilli are highly dynamic and sample multiple orientations over time, marginal protrusions exhibit constrained motion and maintain a vertical orientation. Unexpectedly, we found that marginal microvilli span the junctional space and contact protrusions on neighboring cells, mediated by complexes of CDHR2/CDHR5. FRAP analysis indicated that these transjunctional IMACs are highly stable relative to adhesion complexes between medial microvilli, which explains the restricted motion of protrusions in the marginal zone. Finally, long-term live imaging revealed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions of the cell. Collectively, our findings suggest that nascent microvilli are stabilized by a capture mechanism that is localized to cell margins and enabled by the transjunctional formation of IMACs. These results inform our understanding of how apical specializations are assembled in diverse epithelial systems.
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Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100â days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.
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Células Madre Pluripotentes Inducidas , Enfermedad de Leigh , Células-Madre Neurales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Mutación/genética , Células-Madre Neurales/metabolismo , Organoides/metabolismoRESUMEN
RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.
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Vesículas Extracelulares , Aparato de Golgi , Ceramidas/metabolismo , Retículo Endoplásmico/metabolismo , Vesículas Extracelulares/metabolismo , Aparato de Golgi/metabolismo , Proteínas Serina-Treonina Quinasas , ARN/metabolismoRESUMEN
BACKGROUND: The quantitative analysis of microscope videos often requires instance segmentation and tracking of cellular and subcellular objects. The traditional method consists of two stages: (1) performing instance object segmentation of each frame, and (2) associating objects frame-by-frame. Recently, pixel-embedding-based deep learning approaches these two steps simultaneously as a single stage holistic solution. Pixel-embedding-based learning forces similar feature representation of pixels from the same object, while maximizing the difference of feature representations from different objects. However, such deep learning methods require consistent annotations not only spatially (for segmentation), but also temporally (for tracking). In computer vision, annotated training data with consistent segmentation and tracking is resource intensive, the severity of which is multiplied in microscopy imaging due to (1) dense objects (e.g., overlapping or touching), and (2) high dynamics (e.g., irregular motion and mitosis). Adversarial simulations have provided successful solutions to alleviate the lack of such annotations in dynamics scenes in computer vision, such as using simulated environments (e.g., computer games) to train real-world self-driving systems. METHODS: In this paper, we propose an annotation-free synthetic instance segmentation and tracking (ASIST) method with adversarial simulation and single-stage pixel-embedding based learning. CONTRIBUTION: The contribution of this paper is three-fold: (1) the proposed method aggregates adversarial simulations and single-stage pixel-embedding based deep learning (2) the method is assessed with both the cellular (i.e., HeLa cells); and subcellular (i.e., microvilli) objects; and (3) to the best of our knowledge, this is the first study to explore annotation-free instance segmentation and tracking study for microscope videos. RESULTS: The ASIST method achieved an important step forward, when compared with fully supervised approaches: ASIST shows 7%-11% higher segmentation, detection and tracking performance on microvilli relative to fully supervised methods, and comparable performance on Hela cell videos.
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Procesamiento de Imagen Asistido por Computador , Microscopía , Simulación por Computador , Células HeLa , HumanosRESUMEN
Recently, single-stage embedding based deep learning algorithms gain increasing attention in cell segmentation and tracking. Compared with the traditional "segment-then-associate" two-stage approach, a single-stage algorithm not only simultaneously achieves consistent instance cell segmentation and tracking but also gains superior performance when distinguishing ambiguous pixels on boundaries and overlaps. However, the deployment of an embedding based algorithm is restricted by slow inference speed (e.g., ≈1-2 min per frame). In this study, we propose a novel Faster Mean-shift algorithm, which tackles the computational bottleneck of embedding based cell segmentation and tracking. Different from previous GPU-accelerated fast mean-shift algorithms, a new online seed optimization policy (OSOP) is introduced to adaptively determine the minimal number of seeds, accelerate computation, and save GPU memory. With both embedding simulation and empirical validation via the four cohorts from the ISBI cell tracking challenge, the proposed Faster Mean-shift algorithm achieved 7-10 times speedup compared to the state-of-the-art embedding based cell instance segmentation and tracking algorithm. Our Faster Mean-shift algorithm also achieved the highest computational speed compared to other GPU benchmarks with optimized memory consumption. The Faster Mean-shift is a plug-and-play model, which can be employed on other pixel embedding based clustering inference for medical image analysis. (Plug-and-play model is publicly available: https://github.com/masqm/Faster-Mean-Shift).
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Algoritmos , Rastreo Celular , Análisis por ConglomeradosRESUMEN
The methyltransferase SET domain-containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.
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Actinas , Proteínas de Unión al GTP/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina , Actinas/metabolismo , Citoesqueleto/metabolismo , Lisina/metabolismo , Metilación , Procesamiento Proteico-PostraduccionalRESUMEN
Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.
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Células Epiteliales Alveolares/metabolismo , Fibroblastos/metabolismo , Hiperoxia/genética , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteína Wnt-5a/genética , Animales , Displasia Broncopulmonar , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hiperoxia/metabolismo , Hibridación in Situ , Pulmón/crecimiento & desarrollo , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Microscopía Confocal , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Técnicas de Cultivo de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfonas/farmacología , Proteína Wnt-5a/efectos de los fármacos , Proteína Wnt-5a/metabolismoRESUMEN
Transporting epithelial cells, like those that line the intestinal tract, are specialized for solute processing and uptake. One defining feature is the brush border, an array of microvilli that serves to amplify apical membrane surface area and increase functional capacity. During differentiation, upon exit from stem-cell-containing crypts, enterocytes build thousands of microvilli, each supported by a parallel bundle of actin filaments several microns in length. Given the high concentration of actin residing in mature brush borders, we sought to determine whether enterocytes were resource (i.e., actin monomer) limited in assembling this domain. To examine this possibility, we inhibited Arp2/3, the ubiquitous branched actin nucleator, to increase G-actin availability during brush border assembly. In native intestinal tissues, Arp2/3 inhibition led to increased microvilli length on the surface of crypt, but not villus, enterocytes. In a cell culture model of brush border assembly, Arp2/3 inhibition accelerated the growth and increased the length of microvilli; it also led to a redistribution of F-actin from cortical lateral networks into the brush border. Effects on brush border growth were rescued by treatment with the G-actin sequestering drug, latrunculin A. G-actin binding protein, profilin-1, colocalized in the terminal web with G-actin, and knockdown of this factor compromised brush border growth in a concentration-dependent manner. Finally, the acceleration in brush border assembly induced by Arp2/3 inhibition was abrogated by profilin-1 knockdown. Thus, brush border assembly is limited by G-actin availability, and profilin-1 directs unallocated actin monomers into microvillar core bundles during enterocyte differentiation.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Microvellosidades/metabolismo , Profilinas/metabolismo , Línea Celular Tumoral , Enterocitos/metabolismo , Humanos , Mucosa Intestinal/metabolismoRESUMEN
Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize apical remodeling early in this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at â¼0.2 µm/min. Perturbation with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed ends of motile microvilli, where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher-order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.
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Actinas/metabolismo , Diferenciación Celular , Células Epiteliales/citología , Microvellosidades/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CACO-2 , Cadherinas/metabolismo , Movimiento Celular , Células Epiteliales/metabolismo , Células HEK293 , Humanos , Proteínas de Microfilamentos/metabolismo , Miosinas/metabolismo , PorcinosRESUMEN
Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.
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Actinas/genética , Proteínas de Caenorhabditis elegans/genética , Células Dendríticas/metabolismo , Netrinas/genética , Neuronas/metabolismo , Citoesqueleto de Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de la Membrana/genética , Cadenas Pesadas de Miosina/genética , Proteínas del Tejido Nervioso/genética , Miosina Tipo IIB no Muscular/genéticaRESUMEN
Sepsis costs the healthcare system $23 billion annually and has a mortality rate between 10 and 40%. An early indication of sepsis is the onset of hyperglycemia, which is the result of sepsis-induced insulin resistance in skeletal muscle. Previous investigations have focused on events in the myocyte (e.g., insulin signaling and glucose transport and subsequent metabolism) as the causes for this insulin-resistant state. However, the delivery of insulin to the skeletal muscle is also an important determinant of insulin action. Skeletal muscle microvascular blood flow, which delivers the insulin to the muscle, is known to be decreased during sepsis. Here we test whether the reduced capillary blood flow to skeletal muscle belies the sepsis-induced insulin resistance by reducing insulin delivery to the myocyte. We hypothesize that decreased capillary flow and consequent decrease in insulin delivery is an early event that precedes gross cardiovascular alterations seen with sepsis. This hypothesis was examined in mice treated with either lipopolysaccharide (LPS) or polymicrobial sepsis followed by intravital microscopy of the skeletal muscle microcirculation. We calculated insulin delivery to the myocyte using two independent methods and found that LPS and sepsis rapidly reduce insulin delivery to the skeletal muscle by ~50%; this was driven by decreases in capillary flow velocity and the number of perfused capillaries. Furthermore, the changes in skeletal muscle microcirculation occur before changes in both cardiac output and arterial blood pressure. These data suggest that a rapid reduction in skeletal muscle insulin delivery contributes to the induction of insulin resistance during sepsis.
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Capilares/metabolismo , Hiperglucemia/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Microcirculación , Músculo Esquelético/metabolismo , Sepsis/metabolismo , Animales , Permeabilidad Capilar , Modelos Animales de Enfermedad , Ecocardiografía , Lipopolisacáridos , Ratones , Microvasos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/irrigación sanguíneaRESUMEN
Transporting epithelial cells optimize their morphology for solute uptake by building an apical specialization: a dense array of microvilli that serves to increase membrane surface area. In the intestinal tract, individual cells build thousands of microvilli, which pack tightly to form the brush border. Recent studies implicate adhesion molecule CDHR2 in the regulation of microvillar packing via the formation of adhesion complexes between the tips of adjacent protrusions. To gain insight on how CDHR2 contributes to brush border morphogenesis and enterocyte function under native in vivo conditions, we generated mice lacking CDHR2 expression in the intestinal tract. Although CDHR2 knockout (KO) mice are viable, body weight trends lower and careful examination of tissue, cell, and brush border morphology revealed several perturbations that likely contribute to reduced functional capacity of KO intestine. In the absence of CDHR2, microvilli are significantly shorter, and exhibit disordered packing and a 30% decrease in packing density. These structural perturbations are linked to decreased levels of key solute processing and transporting factors in the brush border. Thus, CDHR2 functions to elongate microvilli and maximize their numbers on the apical surface, which together serve to increase the functional capacity of enterocyte.
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Cadherinas/metabolismo , Microvellosidades/fisiología , Animales , Biomarcadores/metabolismo , Peso Corporal , Cadherinas/genética , Cadherinas/fisiología , Enterocitos/citología , Enterocitos/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones Noqueados , Microvellosidades/ultraestructuraRESUMEN
Wnt/ß-catenin signaling is necessary for normal lung development, and abnormal Wnt signaling contributes to the pathogenesis of both bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF), fibrotic lung diseases that occur during infancy and aging, respectively. Using a library of human normal and diseased human lung samples, we identified a distinct signature of nuclear accumulation of ß-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of ß-catenin phosphorylated at tyrosine 654 in early normal lung development and fibrotic lung diseases BPD and IPF. Furthermore, this signature was recapitulated in murine models of BPD and IPF. Image analysis of immunofluorescence colocalization demonstrated a consistent pattern of elevated nuclear phosphorylated ß-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear ß-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed ß-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of ß-catenin during normal lung development and again in response to lung injury supports the widely held concept that repair of lung injury involves the recapitulation of developmental programs. Furthermore, these observations suggest that ß-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.
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Displasia Broncopulmonar/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , beta Catenina/metabolismo , Células A549 , Adulto , Animales , Animales Recién Nacidos , Proteína Axina/metabolismo , Displasia Broncopulmonar/patología , Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Femenino , Feto/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Fosforilación , Embarazo , Segundo Trimestre del Embarazo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Tirosina/metabolismoRESUMEN
This corrects the article DOI: 10.1038/ncomms10833.
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Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical mediator of type 2 immune response to helminth infection. Here, we present advances in segmentation algorithms and analytical tools for multiplex immunofluorescence (MxIF), a platform that enables iterative staining of over 60 antibodies on a single tissue section. These refinements have enabled a comprehensive analysis of tuft cell number, distribution, and protein expression profiles as a function of anatomical location and physiological perturbations. Based solely on DCLK1 immunoreactivity, tuft cell numbers were similar throughout the mouse small intestine and colon. However, multiple subsets of tuft cells were uncovered when protein coexpression signatures were examined, including two new intestinal tuft cell markers, Hopx and EGFR phosphotyrosine 1068. Furthermore, we identified dynamic changes in tuft cell number, composition, and protein expression associated with fasting and refeeding and after introduction of microbiota to germ-free mice. These studies provide a foundational framework for future studies of intestinal tuft cell regulation and demonstrate the utility of our improved MxIF computational methods and workflow for understanding cellular heterogeneity in complex tissues in normal and disease states.
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High-resolution microscopy has traditionally come at the expense of field of view, resulting in suboptimal interpretation of protein distribution throughout large or complex samples. Likewise, a low-resolution microscopic approach inhibits the ability of researchers to precisely localize proteins of interest at the subcellular level. Until recently, the ability to combine the strengths of these approaches was limited and technically impractical for most laboratories to implement. Continued advances in microscope automation, sophisticated software applications, and modern workstations have enabled expansion of such combinatorial approaches to researchers outside computationally focused fields. Through image stitching, researchers can acquire large field-of-view, multidimensional datasets, at the diffraction limit of high-numerical aperture objectives to effectively map protein distribution in large samples with high precision. Here, we outline a protocol for acquisition of such datasets with the purpose of introducing inexperienced researchers to the methodology of large image stitching using the widely available technology of laser point-scanning confocal microscopy in combination with basic microscope automation and freely available software for post-acquisition processing.
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Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Programas Informáticos , Humanos , Proteínas/metabolismo , Distribución TisularRESUMEN
The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.