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

67 Pages Posted: 26 Jan 2019 Sneak Peek Status: Under Review

See all articles by Anca F. Savulescu

Anca F. Savulescu

University of Cape Town (UCT), Faculty of Health Sciences, Institute for Infectious Disease & Molecular Medicine, Division of Chemical, Systems & Synthetic Biology; CSIR Biosciences, BTRI, Gene Expression and Biophysics Group

Robyn Brackin

Charité - Universitätsmedizin Berlin - Advanced Medical Bioimaging

Emmanuel Bouilhol

University of Bordeaux - Bordeaux Bioinformatics Center; University of Bordeaux - CNRS, LaBRI

Benjamin Dartigues

University of Bordeaux - Bordeaux Bioinformatics Center

Jonathan H. Warrell

Yale University - School of Medicine

Mafalda R. Pimentel

University of Lisbon, Faculty of Medicine, Institute of Molecular Medicine

Stephane Dallongeville

Institut Pasteur, Unite D’Analyse D’Images Biologiques

Jan Schmoranzer

Charité - Universitätsmedizin Berlin - Advanced Medical Bioimaging

Jean-Christophe Olivo-Marin

Institut Pasteur, Unite D’Analyse D’Images Biologiques

Edgar R. Gomes

University of Lisbon, Faculty of Medicine, Institute of Molecular Medicine

Macha Nikolski

University of Bordeaux - Bordeaux Bioinformatics Center; University of Bordeaux - CNRS, LaBRI

Musa Mhlanga

University of Cape Town (UCT) - Division of Chemical, Systems & Synthetic Biology; University of Lisbon - Institute of Molecular Medicine

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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.

Suggested Citation

Savulescu, Anca F. and Brackin, Robyn and Bouilhol, Emmanuel and Dartigues, Benjamin and Warrell, Jonathan H. and Pimentel, Mafalda R. and Dallongeville, Stephane and Schmoranzer, Jan and Olivo-Marin, Jean-Christophe and Gomes, Edgar R. and Nikolski, Macha and Mhlanga, Musa, DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution (January 26, 2019). Available at SSRN: https://ssrn.com/abstract=3323373 or http://dx.doi.org/10.2139/ssrn.3323373
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Anca F. Savulescu

University of Cape Town (UCT), Faculty of Health Sciences, Institute for Infectious Disease & Molecular Medicine, Division of Chemical, Systems & Synthetic Biology

Cape Town
South Africa

CSIR Biosciences, BTRI, Gene Expression and Biophysics Group

Pretoria
South Africa

Robyn Brackin

Charité - Universitätsmedizin Berlin - Advanced Medical Bioimaging

Berlin
Germany

Emmanuel Bouilhol

University of Bordeaux - Bordeaux Bioinformatics Center

Bordeaux
France

University of Bordeaux - CNRS, LaBRI

Talence
France

Benjamin Dartigues

University of Bordeaux - Bordeaux Bioinformatics Center

Bordeaux
France

Jonathan H. Warrell

Yale University - School of Medicine

333 Cedar Street
New Haven, CT 06520-8034
United States

Mafalda R. Pimentel

University of Lisbon, Faculty of Medicine, Institute of Molecular Medicine

R. Branca Edmée Marques
Lisbon, 1600-276
Portugal

Stephane Dallongeville

Institut Pasteur, Unite D’Analyse D’Images Biologiques

Paris
France

Jan Schmoranzer

Charité - Universitätsmedizin Berlin - Advanced Medical Bioimaging

Berlin
Germany

Jean-Christophe Olivo-Marin

Institut Pasteur, Unite D’Analyse D’Images Biologiques

Paris
France

Edgar R. Gomes

University of Lisbon, Faculty of Medicine, Institute of Molecular Medicine ( email )

R. Branca Edmée Marques
Lisbon, 1600-276
Portugal

Macha Nikolski

University of Bordeaux - Bordeaux Bioinformatics Center

Bordeaux
France

University of Bordeaux - CNRS, LaBRI

Talence
France

Musa Mhlanga (Contact Author)

University of Cape Town (UCT) - Division of Chemical, Systems & Synthetic Biology ( email )

Cape Town
South Africa

University of Lisbon - Institute of Molecular Medicine ( email )

R. Branca Edmée Marques
Lisbon, 1600-276
Portugal

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