Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

Dieuwke L. Marvin; Li You; Laura Bornes; Maarten van Dinther; Niek Peters; Hao Dang; Sarah K. Hakuno; Marten Hornsveld; Onno Kranenburg; Jacco van Rheenen; Jos H.T. Rohling; Miao Ping Chien; Peter Ten Dijke; Laila Ritsma

doi:10.3390/cancers14102508

Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

Dieuwke L. Marvin, Li You, Laura Bornes, Maarten van Dinther, Niek Peters, Hao Dang, Sarah K. Hakuno, Marten Hornsveld, Onno Kranenburg, Jacco van Rheenen, Jos H.T. Rohling, Miao Ping Chien, Peter Ten Dijke^*, Laila Ritsma

^*Corresponding author for this work

Molecular Genetics

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

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Abstract

Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

Original language	English
Article number	2508
Journal	Cancers
Volume	14
Issue number	10
DOIs	https://doi.org/10.3390/cancers14102508
Publication status	Published - 19 May 2022

Bibliographical note

Funding Information:
Funding: This research was supported by a Gisela Thier fellowship to L.R. from the Leiden University Medical Center (LUMC), a Veni grant to L.R. from the Netherlands Organisation for Scientific Research (NWO) (016.176.081), a subsidy from the Leids Universiteits Fonds to L.R. (LUF) (CWB 7204), ZonMW grant (09120012010061) (P.t.D) and the Doctor Josef Steiner Foundation (J.v.R.). L.R., P.t.D, and J.v.R. were supported by a subsidy from the Cancer Genomics Center Netherlands (CGC.NL).

Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

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title = "Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells",

abstract = "Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.",

author = "Marvin, {Dieuwke L.} and Li You and Laura Bornes and {van Dinther}, Maarten and Niek Peters and Hao Dang and Hakuno, {Sarah K.} and Marten Hornsveld and Onno Kranenburg and {van Rheenen}, Jacco and Rohling, {Jos H.T.} and Chien, {Miao Ping} and {Ten Dijke}, Peter and Laila Ritsma",

note = "Funding Information: Funding: This research was supported by a Gisela Thier fellowship to L.R. from the Leiden University Medical Center (LUMC), a Veni grant to L.R. from the Netherlands Organisation for Scientific Research (NWO) (016.176.081), a subsidy from the Leids Universiteits Fonds to L.R. (LUF) (CWB 7204), ZonMW grant (09120012010061) (P.t.D) and the Doctor Josef Steiner Foundation (J.v.R.). L.R., P.t.D, and J.v.R. were supported by a subsidy from the Cancer Genomics Center Netherlands (CGC.NL). Publisher Copyright: {\textcopyright} 2022 by the authors. Licensee MDPI, Basel, Switzerland.",

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AU - Marvin, Dieuwke L.

AU - You, Li

AU - Bornes, Laura

AU - van Dinther, Maarten

AU - Peters, Niek

AU - Dang, Hao

AU - Hakuno, Sarah K.

AU - Hornsveld, Marten

AU - Kranenburg, Onno

AU - van Rheenen, Jacco

AU - Rohling, Jos H.T.

AU - Chien, Miao Ping

AU - Ten Dijke, Peter

AU - Ritsma, Laila

N1 - Funding Information: Funding: This research was supported by a Gisela Thier fellowship to L.R. from the Leiden University Medical Center (LUMC), a Veni grant to L.R. from the Netherlands Organisation for Scientific Research (NWO) (016.176.081), a subsidy from the Leids Universiteits Fonds to L.R. (LUF) (CWB 7204), ZonMW grant (09120012010061) (P.t.D) and the Doctor Josef Steiner Foundation (J.v.R.). L.R., P.t.D, and J.v.R. were supported by a subsidy from the Cancer Genomics Center Netherlands (CGC.NL). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

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N2 - Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

AB - Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

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DO - 10.3390/cancers14102508

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JO - Cancers

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ER -

Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

Abstract

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