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1.
Nucleic Acids Res ; 51(9): 4674-4690, 2023 05 22.
Article in English | MEDLINE | ID: mdl-37070176

ABSTRACT

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.


Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic , Transcription Factors , Animals , Mammals , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Nat Commun ; 13(1): 2056, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35440631

ABSTRACT

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.


Subject(s)
Actins , Cilia , Actin Cytoskeleton , Basal Bodies , Cilia/physiology , Cytoskeleton , Humans
3.
Development ; 148(21)2021 11 01.
Article in English | MEDLINE | ID: mdl-34739029

ABSTRACT

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.


Subject(s)
Deep Learning , Embryonic Development/genetics , Phenotype , Animals , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/pathology , Disease Models, Animal , Image Processing, Computer-Assisted , Mice , Microscopy , Mutation , Neural Networks, Computer , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Polycystic Kidney Diseases/embryology , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/pathology , Xenopus Proteins/genetics , Xenopus laevis
4.
Nat Methods ; 16(4): 351, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30804552

ABSTRACT

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.

5.
Science ; 363(6425)2019 01 25.
Article in English | MEDLINE | ID: mdl-30679343

ABSTRACT

The innate immune cell compartment is highly diverse in the healthy central nervous system (CNS), including parenchymal and non-parenchymal macrophages. However, this complexity is increased in inflammatory settings by the recruitment of circulating myeloid cells. It is unclear which disease-specific myeloid subsets exist and what their transcriptional profiles and dynamics during CNS pathology are. Combining deep single-cell transcriptome analysis, fate mapping, in vivo imaging, clonal analysis, and transgenic mouse lines, we comprehensively characterized unappreciated myeloid subsets in several CNS compartments during neuroinflammation. During inflammation, CNS macrophage subsets undergo self-renewal, and random proliferation shifts toward clonal expansion. Last, functional studies demonstrated that endogenous CNS tissue macrophages are redundant for antigen presentation. Our results highlight myeloid cell diversity and provide insights into the brain's innate immune system.


Subject(s)
Central Nervous System/immunology , Immunity, Innate , Inflammation/immunology , Macrophages/cytology , Myeloid Cells/cytology , Animals , Antigen Presentation , Brain/immunology , Dendritic Cells/cytology , Encephalomyelitis, Autoimmune, Experimental/immunology , Histocompatibility Antigens Class II/immunology , Homeostasis , Macrophages/immunology , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/cytology , Myeloid Cells/immunology , Sequence Analysis, RNA , Single-Cell Analysis , T-Lymphocytes/immunology
6.
Nat Methods ; 16(1): 67-70, 2019 01.
Article in English | MEDLINE | ID: mdl-30559429

ABSTRACT

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.


Subject(s)
Cell Count , Deep Learning , Cloud Computing , Neural Networks, Computer , Software Design
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