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      Protocol for ex vivo time lapse imaging of Drosophila melanogaster cytonemes

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          Summary

          This protocol describes how to image time and spatially resolved time lapses of Drosophila air sac primordium (ASP) cytonemes in ex vivo cultures of wing imaginal discs. It describes how to manually measure the length of cytonemes using custom-made FIJI/ImageJ tools, and to analyze data using R/R-Studios pipeline. It can also be used for studies of cell division, organelle localization, and protein trafficking as well as other cellular materials that can be fluorescently tagged and imaged with minimal phototoxicity.

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          Highlights

          • •

            This protocol enables imaging of air sac primordium cytonemes dynamics for 4–5 h

          • •

            ImageJ/FIJI Cytoneme Analysis tools provided for measuring cytoneme dynamics

          • •

            RStudios pipeline provided for data processing of cytoneme dynamics measurements

          Abstract

          This protocol describes how to image time and spatially resolved time lapses of Drosophila air sac primordium (ASP) cytonemes in ex vivo cultures of wing imaginal discs. It describes how to manually measure the length of cytonemes using custom-made FIJI/ImageJ tools, and to analyze data using a R/R-Studios pipeline. It can also be used for studies of cell division, organelle localization, and protein trafficking as well as other cellular materials that can be fluorescently tagged and imaged with minimal phototoxicity.

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          Most cited references11

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          Fiji: an open-source platform for biological-image analysis.

          Johannes Schindelin, Ignacio Arganda-Carreras, Erwin Frise … (2019)
          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.
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            Democratising deep learning for microscopy with ZeroCostDL4Mic

            Lucas von Chamier, Romain F. Laine, Johanna Jukkala … (2021)
            Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes. Deep learning methods show great promise for the analysis of microscopy images but there is currently an accessibility barrier to many users. Here the authors report a convenient entry-level deep learning platform that can be used at no cost: ZeroCostDL4Mic.
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              Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein.

              Stephanie S. Liu, Hai-Zheng Huang, Thomas B. Kornberg … (2014)
              Decapentaplegic (Dpp), a Drosophila morphogen signaling protein, transfers directly at synapses made at sites of contact between cells that produce Dpp and cytonemes that extend from recipient cells. The Dpp that cytonemes receive moves together with activated receptors toward the recipient cell body in motile puncta. Genetic loss-of-function conditions for diaphanous, shibire, neuroglian, and capricious perturbed cytonemes by reducing their number or only the synapses they make with cells they target, and reduced cytoneme-mediated transport of Dpp and Dpp signaling. These experiments provide direct evidence that cells use cytonemes to exchange signaling proteins, that cytoneme-based exchange is essential for signaling and normal development, and that morphogen distribution and signaling can be contact-dependent, requiring cytoneme synapses.
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                Author and article information

                Contributors
                Guilherme O. Barbosa
                Thomas B. Kornberg
                Journal
                Journal ID (nlm-ta): STAR Protoc
                Journal ID (iso-abbrev): STAR Protoc
                Title: STAR Protocols
                Publisher: Elsevier
                ISSN (Electronic): 2666-1667
                Publication date PMC-release: 29 January 2022
                Publication date Collection: 18 March 2022
                Publication date (Electronic): 29 January 2022
                Volume: 3
                Issue: 1
                Electronic Location Identifier: 101138
                Affiliations
                [1 ]Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA
                Author notes
                [∗ ]Corresponding author gbarbosa.bio@ 123456gmail.com
                [∗∗ ]Corresponding author tkornberg@ 123456ucsf.edu
                [2]

                Technical contact

                [3]

                Lead contact

                Article
                Publisher Item ID: S2666-1667(22)00018-1 Publisher ID: 101138
                DOI: 10.1016/j.xpro.2022.101138
                PMC ID: 8810567
                SO-VID: cfb1522a-7f6f-41fb-8a77-8500001ab1b7
                Copyright © © 2022 The Authors
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Categories
                Subject: Protocol

                Keywords: cell biology,developmental biology,microscopy,model organisms
                Data availability:
                Keywords: cell biology, developmental biology, microscopy, model organisms

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