Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH

Anca F. Savulescu; Robyn Brackin; Emmanuel Bouilhol; Benjamin Dartigues; Jonathan H. Warrell; Mafalda R. Pimentel; Nicolas Beaume; Isabela C. Fortunato; Stephane Dallongeville; Mikaël Boulle; Hayssam Soueidan; Fabrice Agou; Jan Schmoranzer; Jean Christophe Olivo-Marin; Cláudio A. Franco; Edgar R. Gomes; Macha Nikolski; Musa M. Mhlanga

doi:10.1016/j.crmeth.2021.100068

Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH

Anca F. Savulescu^*, Robyn Brackin, Emmanuel Bouilhol, Benjamin Dartigues, Jonathan H. Warrell, Mafalda R. Pimentel, Nicolas Beaume, Isabela C. Fortunato, Stephane Dallongeville, Mikaël Boulle, Hayssam Soueidan, Fabrice Agou, Jan Schmoranzer, Jean Christophe Olivo-Marin, Cláudio A. Franco, Edgar R. Gomes, Macha Nikolski, Musa M. Mhlanga

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

14 Citations (Scopus)

Abstract

Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

Original language	English
Article number	100068
Journal	Cell Reports Methods
Volume	1
Issue number	5
DOIs	https://doi.org/10.1016/j.crmeth.2021.100068
Publication status	Published - 27 Sept 2021
Externally published	Yes

Keywords

Image analysis
Microfabricated patterns
Ripley's K
RNA subcellular localization
Single-molecule FISH

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Savulescu, A. F., Brackin, R., Bouilhol, E., Dartigues, B., Warrell, J. H., Pimentel, M. R., Beaume, N., Fortunato, I. C., Dallongeville, S., Boulle, M., Soueidan, H., Agou, F., Schmoranzer, J., Olivo-Marin, J. C., Franco, C. A., Gomes, E. R., Nikolski, M., & Mhlanga, M. M. (2021). Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH. Cell Reports Methods, 1(5), Article 100068. https://doi.org/10.1016/j.crmeth.2021.100068

@article{bbd1f5e2237a4d959a4e50fb0ab42028,

title = "Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH",

abstract = "Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.",

keywords = "Image analysis, Microfabricated patterns, Ripley's K, RNA subcellular localization, Single-molecule FISH",

author = "Savulescu, {Anca F.} and Robyn Brackin and Emmanuel Bouilhol and Benjamin Dartigues and Warrell, {Jonathan H.} and Pimentel, {Mafalda R.} and Nicolas Beaume and Fortunato, {Isabela C.} and Stephane Dallongeville and Mika{\"e}l Boulle and Hayssam Soueidan and Fabrice Agou and Jan Schmoranzer and Olivo-Marin, {Jean Christophe} and Franco, {Cl{\'a}udio A.} and Gomes, {Edgar R.} and Macha Nikolski and Mhlanga, {Musa M.}",

note = "Funding Information: We thank all members of the Gene Expression and Biophysics Laboratory (Mhlanga Lab). We thank N. Crosetto, and members of the L. Pelkmans lab, members of the J.-C. Olivo-Marin lab, K. Schauer, and members of the B. Goud lab for comments on the manuscript. A.F.S. is a recipient of a postdoctoral fellowship from the Claude Leon Foundation , South Africa. This research has been supported by the following grants, all to M.M.M. V2YGE81 (to A.F.S.) and PG-V2KYPO7 , TA 2011 011 from the Council for Industrial and Scientific Research ( CSIR ) (South Africa) and by a grant from the Emerging Research Area Program of The Department of Science and Technology (South Africa) Department of Science & Technology Center of Competence Grant , SA Medical Research Council SHIP grant, and CSIR Parliamentary Grant . M.M.M. and M.N. are Chan Zuckerberg Investigators of the Chan Zuckerberg Initiative – Human Cell Atlas program. Funding Information: We thank all members of the Gene Expression and Biophysics Laboratory (Mhlanga Lab). We thank N. Crosetto, and members of the L. Pelkmans lab, members of the J.-C. Olivo-Marin lab, K. Schauer, and members of the B. Goud lab for comments on the manuscript. A.F.S. is a recipient of a postdoctoral fellowship from the Claude Leon Foundation, South Africa. This research has been supported by the following grants, all to M.M.M. V2YGE81 (to A.F.S.) and PG-V2KYPO7, TA 2011 011 from the Council for Industrial and Scientific Research (CSIR) (South Africa) and by a grant from the Emerging Research Area Program of The Department of Science and Technology (South Africa) Department of Science & Technology Center of Competence Grant, SA Medical Research Council SHIP grant, and CSIR Parliamentary Grant. M.M.M. and M.N. are Chan Zuckerberg Investigators of the Chan Zuckerberg Initiative – Human Cell Atlas program. Conceptualization, M.M.M. M.N. and A.F.S.; methodology, A.F.S. R.B. J.H.W. M.R.P. N.B. I.C.F. S.D. M.B. M.N. E.B. B.D. F.A. C.A.F. E.R.G. and J.-C.O.-M.; software, M.N. E.B. B.D. H.S. J.-C.O.-M. J.H.W. M.B. and S.D.; formal analysis, M.N. E.B. and B.D.; investigation, A.F.S. R.B. M.R.P. J.S. and I.C.F.; data curation, A.F.S. M.N. E.B. and B.D.; writing – original draft, A.F.S. R.B. J.H.W. M.N. M.R.P. and M.M.M.; writing – review and editing, M.N. M.M.M. and A.F.S.; supervision, M.M.M. C.A.F. E.R.G. and M.N.; project administration, M.M.M. M.N. and A.F.S. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. The author list of this paper includes contributors from the location where the research was conducted who participated in the data collection, design, analysis, and/or interpretation of the work. Publisher Copyright: {\textcopyright} 2022 The Authors",

year = "2021",

month = sep,

day = "27",

doi = "10.1016/j.crmeth.2021.100068",

language = "English",

volume = "1",

journal = "Cell Reports Methods",

issn = "2667-2375",

publisher = "Cell Press",

number = "5",

}

Savulescu, AF, Brackin, R, Bouilhol, E, Dartigues, B, Warrell, JH, Pimentel, MR, Beaume, N, Fortunato, IC, Dallongeville, S, Boulle, M, Soueidan, H, Agou, F, Schmoranzer, J, Olivo-Marin, JC, Franco, CA, Gomes, ER, Nikolski, M & Mhlanga, MM 2021, 'Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH', Cell Reports Methods, vol. 1, no. 5, 100068. https://doi.org/10.1016/j.crmeth.2021.100068

TY - JOUR

T1 - Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH

AU - Savulescu, Anca F.

AU - Brackin, Robyn

AU - Bouilhol, Emmanuel

AU - Dartigues, Benjamin

AU - Warrell, Jonathan H.

AU - Pimentel, Mafalda R.

AU - Beaume, Nicolas

AU - Fortunato, Isabela C.

AU - Dallongeville, Stephane

AU - Boulle, Mikaël

AU - Soueidan, Hayssam

AU - Agou, Fabrice

AU - Schmoranzer, Jan

AU - Olivo-Marin, Jean Christophe

AU - Franco, Cláudio A.

AU - Gomes, Edgar R.

AU - Nikolski, Macha

AU - Mhlanga, Musa M.

N1 - Funding Information: We thank all members of the Gene Expression and Biophysics Laboratory (Mhlanga Lab). We thank N. Crosetto, and members of the L. Pelkmans lab, members of the J.-C. Olivo-Marin lab, K. Schauer, and members of the B. Goud lab for comments on the manuscript. A.F.S. is a recipient of a postdoctoral fellowship from the Claude Leon Foundation , South Africa. This research has been supported by the following grants, all to M.M.M. V2YGE81 (to A.F.S.) and PG-V2KYPO7 , TA 2011 011 from the Council for Industrial and Scientific Research ( CSIR ) (South Africa) and by a grant from the Emerging Research Area Program of The Department of Science and Technology (South Africa) Department of Science & Technology Center of Competence Grant , SA Medical Research Council SHIP grant, and CSIR Parliamentary Grant . M.M.M. and M.N. are Chan Zuckerberg Investigators of the Chan Zuckerberg Initiative – Human Cell Atlas program. Funding Information: We thank all members of the Gene Expression and Biophysics Laboratory (Mhlanga Lab). We thank N. Crosetto, and members of the L. Pelkmans lab, members of the J.-C. Olivo-Marin lab, K. Schauer, and members of the B. Goud lab for comments on the manuscript. A.F.S. is a recipient of a postdoctoral fellowship from the Claude Leon Foundation, South Africa. This research has been supported by the following grants, all to M.M.M. V2YGE81 (to A.F.S.) and PG-V2KYPO7, TA 2011 011 from the Council for Industrial and Scientific Research (CSIR) (South Africa) and by a grant from the Emerging Research Area Program of The Department of Science and Technology (South Africa) Department of Science & Technology Center of Competence Grant, SA Medical Research Council SHIP grant, and CSIR Parliamentary Grant. M.M.M. and M.N. are Chan Zuckerberg Investigators of the Chan Zuckerberg Initiative – Human Cell Atlas program. Conceptualization, M.M.M. M.N. and A.F.S.; methodology, A.F.S. R.B. J.H.W. M.R.P. N.B. I.C.F. S.D. M.B. M.N. E.B. B.D. F.A. C.A.F. E.R.G. and J.-C.O.-M.; software, M.N. E.B. B.D. H.S. J.-C.O.-M. J.H.W. M.B. and S.D.; formal analysis, M.N. E.B. and B.D.; investigation, A.F.S. R.B. M.R.P. J.S. and I.C.F.; data curation, A.F.S. M.N. E.B. and B.D.; writing – original draft, A.F.S. R.B. J.H.W. M.N. M.R.P. and M.M.M.; writing – review and editing, M.N. M.M.M. and A.F.S.; supervision, M.M.M. C.A.F. E.R.G. and M.N.; project administration, M.M.M. M.N. and A.F.S. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. The author list of this paper includes contributors from the location where the research was conducted who participated in the data collection, design, analysis, and/or interpretation of the work. Publisher Copyright: © 2022 The Authors

PY - 2021/9/27

Y1 - 2021/9/27

N2 - Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

AB - Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

KW - Image analysis

KW - Microfabricated patterns

KW - Ripley's K

KW - RNA subcellular localization

KW - Single-molecule FISH

UR - http://www.scopus.com/inward/record.url?scp=85122683200&partnerID=8YFLogxK

U2 - 10.1016/j.crmeth.2021.100068

DO - 10.1016/j.crmeth.2021.100068

M3 - Article

C2 - 35474672

SN - 2667-2375

VL - 1

JO - Cell Reports Methods

JF - Cell Reports Methods

IS - 5

M1 - 100068

ER -

Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH

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Keywords

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