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1.
Development ; 151(4)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38240380

RESUMO

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.


Assuntos
Músculo Esquelético , Músculos Oculomotores , Camundongos , Animais , Músculo Esquelético/metabolismo , Músculos Oculomotores/metabolismo , Camundongos Endogâmicos C57BL , Proliferação de Células , Células-Tronco
2.
PLoS Biol ; 22(8): e3002740, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39116189

RESUMO

In life sciences, tracking objects from movies enables researchers to quantify the behavior of single particles, organelles, bacteria, cells, and even whole animals. While numerous tools now allow automated tracking from video, a significant challenge persists in compiling, analyzing, and exploring the large datasets generated by these approaches. Here, we introduce CellTracksColab, a platform tailored to simplify the exploration and analysis of cell tracking data. CellTracksColab facilitates the compiling and analysis of results across multiple fields of view, conditions, and repeats, ensuring a holistic dataset overview. CellTracksColab also harnesses the power of high-dimensional data reduction and clustering, enabling researchers to identify distinct behavioral patterns and trends without bias. Finally, CellTracksColab also includes specialized analysis modules enabling spatial analyses (clustering, proximity to specific regions of interest). We demonstrate CellTracksColab capabilities with 3 use cases, including T cells and cancer cell migration, as well as filopodia dynamics. CellTracksColab is available for the broader scientific community at https://github.com/CellMigrationLab/CellTracksColab.


Assuntos
Movimento Celular , Rastreamento de Células , Software , Rastreamento de Células/métodos , Humanos , Animais , Processamento de Imagem Assistida por Computador/métodos , Pseudópodes/fisiologia , Linfócitos T , Camundongos
3.
Nat Methods ; 19(7): 829-832, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35654950

RESUMO

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.


Assuntos
Algoritmos , Software , Processamento de Imagem Assistida por Computador/métodos
4.
J Microsc ; 294(3): 276-294, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38656474

RESUMO

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.


Assuntos
Disciplinas das Ciências Biológicas , Disciplinas das Ciências Biológicas/métodos
5.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34599102

RESUMO

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.


Assuntos
Bacteriocinas/metabolismo , Membrana Celular/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/metabolismo , Trifosfato de Adenosina/metabolismo , Citoplasma/metabolismo
6.
BMC Biol ; 20(1): 183, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35999534

RESUMO

BACKGROUND: Efficient tools allowing the extraction of 2D surfaces from 3D-microscopy data are essential for studies aiming to decipher the complex cellular choreography through which epithelium morphogenesis takes place during development. Most existing methods allow for the extraction of a single and smooth manifold of sufficiently high signal intensity and contrast, and usually fail when the surface of interest has a rough topography or when its localization is hampered by other surrounding structures of higher contrast. Multiple surface segmentation entails laborious manual annotations of the various surfaces separately. RESULTS: As automating this task is critical in studies involving tissue-tissue or tissue-matrix interaction, we developed the Zellige software, which allows the extraction of a non-prescribed number of surfaces of varying inclination, contrast, and texture from a 3D image. The tool requires the adjustment of a small set of control parameters, for which we provide an intuitive interface implemented as a Fiji plugin. CONCLUSIONS: As a proof of principle of the versatility of Zellige, we demonstrate its performance and robustness on synthetic images and on four different types of biological samples, covering a wide range of biological contexts.


Assuntos
Algoritmos , Microscopia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Software
8.
BMC Biol ; 19(1): 136, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215263

RESUMO

BACKGROUND: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. RESULTS: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. CONCLUSIONS: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.


Assuntos
Imageamento Tridimensional , Microscopia
9.
Cell Microbiol ; 22(5): e13166, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31957253

RESUMO

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.


Assuntos
Citoesqueleto de Actina/metabolismo , Imunidade Adaptativa , Disenteria Bacilar/imunologia , Transporte Proteico/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Shigella/metabolismo , Actinas , Linhagem Celular , Complexo de Golgi , Humanos , Sinapses Imunológicas , Shigella/genética , Linfócitos T/imunologia , Sistemas de Secreção Tipo III/metabolismo
10.
Methods ; 115: 80-90, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27713081

RESUMO

We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results. The utility of TrackMate is further enhanced through its ability to be readily customized to meet specific tracking problems. TrackMate is an extensible platform where developers can easily write their own detection, particle linking, visualization or analysis algorithms within the TrackMate environment. This evolving framework provides researchers with the opportunity to quickly develop and optimize new algorithms based on existing TrackMate modules without the need of having to write de novo user interfaces, including visualization, analysis and exporting tools. The current capabilities of TrackMate are presented in the context of three different biological problems. First, we perform Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs its early development. Our TrackMate-based lineage analysis indicates the lack of a cell-specific light-sensitive mechanism. Second, we investigate the recruitment of NEMO (NF-κB essential modulator) clusters in fibroblasts after stimulation by the cytokine IL-1 and show that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, we validate the use of TrackMate for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells.


Assuntos
Rastreamento de Células/métodos , Embrião não Mamífero/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Análise de Célula Única/métodos , Software , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Algoritmos , Animais , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Caenorhabditis elegans , Rastreamento de Células/estatística & dados numéricos , Clatrina/genética , Clatrina/metabolismo , Embrião não Mamífero/metabolismo , Endocitose , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Análise de Célula Única/estatística & dados numéricos
11.
Proc Natl Acad Sci U S A ; 112(25): E3282-90, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26056271

RESUMO

Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent.


Assuntos
Colo/microbiologia , Disenteria Bacilar/patologia , Shigella flexneri/patogenicidade , Animais , Cobaias , Humanos , Mucosa Intestinal/microbiologia
12.
Development ; 141(8): 1726-36, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24715462

RESUMO

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Medula Espinal/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Interneurônios/citologia , Interneurônios/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Medula Espinal/embriologia , Células-Tronco/metabolismo
13.
Nat Methods ; 11(3): 281-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24441936

RESUMO

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.


Assuntos
Interpretação de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Interpretação de Imagem Assistida por Computador/normas , Microscopia de Fluorescência/normas
14.
Nature ; 476(7361): 462-6, 2011 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-21822289

RESUMO

Cytokinesis, the physical separation of daughter cells at the end of mitosis, requires precise regulation of the mechanical properties of the cell periphery. Although studies of cytokinetic mechanics mostly focus on the equatorial constriction ring, a contractile actomyosin cortex is also present at the poles of dividing cells. Whether polar forces influence cytokinetic cell shape and furrow positioning remains an open question. Here we demonstrate that the polar cortex makes cytokinesis inherently unstable. We show that limited asymmetric polar contractions occur during cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations, resulting in furrow displacement and aneuploidy. A theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. We further propose that membrane blebs, which commonly form at the poles of dividing cells and whose role in cytokinesis has long been enigmatic, stabilize cell shape by acting as valves releasing cortical contractility. Our findings reveal an inherent instability in the shape of the dividing cell and unveil a novel, spindle-independent mechanism ensuring the stability of cleavage furrow positioning.


Assuntos
Actomiosina/metabolismo , Forma Celular/fisiologia , Citocinese/fisiologia , Amidas/farmacologia , Aneuploidia , Linhagem Celular , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Células HeLa , Humanos , Modelos Biológicos , Piridinas/farmacologia
15.
Nat Methods ; 9(7): 676-82, 2012 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-22743772

RESUMO

Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.


Assuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Software , Algoritmos , Animais , Encéfalo/ultraestrutura , Drosophila melanogaster/ultraestrutura , Aumento da Imagem/métodos , Imageamento Tridimensional/métodos , Disseminação de Informação , Design de Software
16.
Methods ; 66(2): 353-61, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24045025

RESUMO

Energy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting.


Assuntos
Transferência de Energia , Algoritmos , Escherichia coli , Corantes Fluorescentes/química , Klebsiella pneumoniae , Luciferases de Renilla/química , Pontos Quânticos/química , Espectrometria de Fluorescência
17.
Bioessays ; 35(5): 472-81, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23450621

RESUMO

When a cell divides, it produces two daughter cells initially connected by a cytokinesis bridge, which is eventually cut through abscission. One of the two daughter cells inherits a bridge "remnant", which has been proposed to be degraded by autophagy. The fate and function of remnants is attracting increasing attention, as their accumulation appears to influence proliferation versus differentiation of the daughter cells. Here, we present a simple model for bridge and remnant turnover in a dynamic cell population. We demonstrate that remnant proportions depend on the ratio of remnant and bridge lifetimes to the cell population doubling time. Our results yield new alternative interpretations for published experimental data, leading us to believe that autophagy-independent pathways for remnant degradation may exist. In addition, using the model, we determined experimentally inaccessible parameters such as remnant lifetime. Our model proves to be a useful tool for studying bridge and remnant populations.


Assuntos
Citocinese , Fibroblastos/citologia , Modelos Biológicos , Animais , Autofagia , Diferenciação Celular , Membrana Celular/metabolismo , Proliferação de Células , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Microtúbulos/metabolismo , Projetos de Pesquisa , Fatores de Tempo
18.
Nat Commun ; 15(1): 4175, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755132

RESUMO

Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat.


Assuntos
Antituberculosos , Mycobacterium tuberculosis , Análise de Célula Única , Tuberculose , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Análise de Célula Única/métodos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Microfluídica/métodos , Fenótipo , Descoberta de Drogas/métodos , Sinergismo Farmacológico
19.
J Cell Sci ; 124(Pt 18): 3053-9, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21868367

RESUMO

The early control of potentially invading microbes by our immune system primarily depends on its main professional phagocytes - macrophages and neutrophils. Although the different functions of these two cell types have been extensively studied, little is known about their respective contributions to the initial control of invading microorganisms before the onset of adaptive immune responses. The naturally translucent zebrafish larva has recently emerged as a powerful model vertebrate in which to visualise the dynamic interactions between leukocytes and microbes in vivo. Using high-resolution live imaging, we found that whereas macrophages efficiently engulf bacteria from blood or fluid-filled body cavities, neutrophils barely do so. By contrast, neutrophils very efficiently sweep up surface-associated, but not fluid-borne, bacteria. Thus the physical presentation of unopsonised microbes is a crucial determinant of neutrophil phagocytic ability. Neutrophils engulf microbes only as they move over them, in a 'vacuum-cleaner' type of behaviour. This context-dependent nature of phagocytosis by neutrophils should be of particular relevance to human infectious diseases, especially for the early phase of encounter with microbes new to the host.


Assuntos
Líquidos Corporais/metabolismo , Escherichia coli/imunologia , Macrófagos/metabolismo , Neutrófilos/metabolismo , Fagocitose , Animais , Aderência Bacteriana/imunologia , Células Cultivadas , Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata , Larva , Macrófagos/imunologia , Macrófagos/patologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Fagocitose/imunologia , Imagem com Lapso de Tempo , Peixe-Zebra
20.
J Cell Biol ; 222(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36880553

RESUMO

Single-particle tracking microscopy is a powerful technique to investigate how proteins dynamically interact with their environment in live cells. However, the analysis of tracks is confounded by noisy molecule localization, short tracks, and rapid transitions between different motion states, notably between immobile and diffusive states. Here, we propose a probabilistic method termed ExTrack that uses the full spatio-temporal information of tracks to extract global model parameters, to calculate state probabilities at every time point, to reveal distributions of state durations, and to refine the positions of bound molecules. ExTrack works for a wide range of diffusion coefficients and transition rates, even if experimental data deviate from model assumptions. We demonstrate its capacity by applying it to slowly diffusing and rapidly transitioning bacterial envelope proteins. ExTrack greatly increases the regime of computationally analyzable noisy single-particle tracks. The ExTrack package is available in ImageJ and Python.


Assuntos
Proteínas de Bactérias , Microscopia , Difusão , Cinética
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