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DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution

Anca F. Savulescu, Robyn Brackin, Emmanuel Bouilhol, Benjamin Dartigues, Jonathan H. Warrell, Mafalda R. Pimentel, Stephane Dallongeville, Jan Schmoranzer, Jean-Christophe Olivo-Marin, Edgar R. Gomes, Macha Nikolski, Musa M. Mhlanga
doi: https://doi.org/10.1101/536383
Anca F. Savulescu
University of Cape Town;
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Robyn Brackin
Charite Universitatsmedizin Berlin;
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Emmanuel Bouilhol
Universite de Bordeaux;
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Benjamin Dartigues
Universite de Bordeaux;
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Jonathan H. Warrell
Yale School of Medicine;
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Mafalda R. Pimentel
Universidade de Lisboa;
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Stephane Dallongeville
Institut Pasteur
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Jan Schmoranzer
Charite Universitatsmedizin Berlin;
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Jean-Christophe Olivo-Marin
Institut Pasteur
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Edgar R. Gomes
Universidade de Lisboa;
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Macha Nikolski
Universite de Bordeaux;
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Musa M. Mhlanga
University of Cape Town;
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  • For correspondence: musa@mhlangalab.org
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Abstract

Advances in single cell RNA sequencing have allowed for the identification and characterization of cellular subtypes based on quantification of the number of transcripts in each cell. However, cells may differ not only in the number of mRNA transcripts that they exhibit, but also in their spatial and temporal distribution, intrinsic to the definition of their cellular state. Here we describe DypFISH, an approach to quantitatively investigate the spatial and temporal subcellular localization of RNA and protein, by combining micropatterning of cells with fluorescence microscopy at high resolution. We introduce a range of analytical techniques for quantitatively interrogating single molecule RNA FISH data in combination with protein immunolabeling over time. Strikingly, our results show that constraining cellular architecture reduces variation in subcellular mRNA and protein distributions, allowing the characterization of their localization and dynamics with high reproducibility. Many tissues contain cells that exist in similar constrained architectures. Thus DypFISH reveals reproducible patterns of clustering, strong correlative influences of mRNA-protein localization on MTOC orientation when they are present and interdependent dynamics globally and at specific subcellular locations which can be extended to physiological systems.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted January 31, 2019.
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DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution
Anca F. Savulescu, Robyn Brackin, Emmanuel Bouilhol, Benjamin Dartigues, Jonathan H. Warrell, Mafalda R. Pimentel, Stephane Dallongeville, Jan Schmoranzer, Jean-Christophe Olivo-Marin, Edgar R. Gomes, Macha Nikolski, Musa M. Mhlanga
bioRxiv 536383; doi: https://doi.org/10.1101/536383
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DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution
Anca F. Savulescu, Robyn Brackin, Emmanuel Bouilhol, Benjamin Dartigues, Jonathan H. Warrell, Mafalda R. Pimentel, Stephane Dallongeville, Jan Schmoranzer, Jean-Christophe Olivo-Marin, Edgar R. Gomes, Macha Nikolski, Musa M. Mhlanga
bioRxiv 536383; doi: https://doi.org/10.1101/536383

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