RESUMO
U-Net is a generic deep-learning solution for frequently occurring quantification tasks such as cell detection and shape measurements in biomedical image data. We present an ImageJ plugin that enables non-machine-learning experts to analyze their data with U-Net on either a local computer or a remote server/cloud service. The plugin comes with pretrained models for single-cell segmentation and allows for U-Net to be adapted to new tasks on the basis of a few annotated samples.
Assuntos
Contagem de Células , Aprendizado Profundo , Computação em Nuvem , Redes Neurais de Computação , Design de SoftwareRESUMO
In the version of this paper originally published, one of the affiliations for Dominic Mai was incorrect: "Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany" should have been "Life Imaging Center, Center for Biological Systems Analysis, Albert-Ludwigs-University, Freiburg, Germany." This change required some renumbering of subsequent author affiliations. These corrections have been made in the PDF and HTML versions of the article, as well as in any cover sheets for associated Supplementary Information.
RESUMO
We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.
Assuntos
Algoritmos , Rastreamento de Células/métodos , Interpretação de Imagem Assistida por Computador , Benchmarking , Linhagem Celular , HumanosRESUMO
In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells.
Assuntos
Retículo Endoplasmático/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Plantas/genética , Humanos , Proteínas de Plantas/metabolismo , Transdução de SinaisRESUMO
It has long been known that electrical fields (EFs) are able to influence the direction of migrating cells, a process commonly referred to as electrotaxis or galvanotaxis. Most studies have focused on migrating cells equipped with an existing polarity before EF application, making it difficult to delineate EF-specific pathways. Here we study the initial events in front-rear organization of spreading keratinocytes to dissect the molecular requirements for random and EF-controlled polarization. We find that Arp2/3-dependent protrusive forces and Rac1/Cdc42 activity were generally required for both forms of polarization but were dispensable for controlling the direction of EF-controlled polarization. By contrast, we found a crucial role for extracellular pH as well as G protein coupled-receptor (GPCR) or purinergic signaling in the control of directionality. The normal direction of polarization toward the cathode was reverted by lowering extracellular pH. Polarization toward the anode was also seen at neutral pH when GPCR or purinergic signaling was inhibited. However, the stepwise increase of extracellular pH in this scenario led to restoration of cathodal polarization. Overall our work puts forward a model in which the EF uses distinct polarization pathways. The cathodal pathway involves GPCR/purinergic signaling and is dominant over the anodal pathway at neutral pH.
Assuntos
Polaridade Celular/fisiologia , Queratinócitos/citologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Estimulação Elétrica , Eletricidade , Humanos , Concentração de Íons de Hidrogênio , Indóis/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Transdução de SinaisRESUMO
Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Corpos Basais/metabolismo , Drosophila melanogaster , Epiderme/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Xenopus laevis/metabolismo , Proteínas rho de Ligação ao GTPRESUMO
Epiboly is the first coordinated cell movement in most vertebrates and marks the onset of gastrulation. During zebrafish epiboly, enveloping layer (EVL) and deep cells spread over the vegetal yolk mass with a concomitant thinning of the deep cell layer. A prevailing model suggests that deep cell radial intercalations directed towards the EVL would drive deep cell epiboly. To test this model, we have globally recorded 3D cell trajectories for zebrafish blastomeres between sphere and 50% epiboly stages, and developed an image analysis framework to determine intercalation events, intercalation directionality, and migration speed for cells at specific positions within the embryo. This framework uses Voronoi diagrams to compute cell-to-cell contact areas, defines a feature-based spatio-temporal model for intercalation events and fits an anatomical coordinate system to the recorded datasets. We further investigate whether epiboly defects in MZspg mutant embryos devoid of Pou5f1/Oct4 may be caused by changes in intercalation behavior. In wild-type and mutant embryos, intercalations orthogonal to the EVL occur with no directional bias towards or away from the EVL, suggesting that there are no directional cues that would direct intercalations towards the EVL. Further, we find that intercalation direction is independent of the previous intercalation history of individual deep cells, arguing against cues that would program specific intrinsic directed migration behaviors. Our data support a dynamic model in which deep cells during epiboly migrate into space opening between the EVL and the yolk syncytial layer. Genetic programs determining cell motility may control deep cell dynamic behavior and epiboly progress.