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

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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

Keywords

signaling dynamics
signaling heterogeneity
TGF-β signaling
transcriptional reporter

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cancers-14-02508Final published version, 32 MBLicence: CC BY

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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.",

keywords = "signaling dynamics, signaling heterogeneity, TGF-β signaling, transcriptional reporter",

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 Miao-Ping Chien 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.",

year = "2022",

month = may,

day = "19",

doi = "10.3390/cancers14102508",

language = "English",

volume = "14",

journal = "Cancers",

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T1 - Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

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.

PY - 2022/5/19

Y1 - 2022/5/19

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.

KW - signaling dynamics

KW - signaling heterogeneity

KW - TGF-β signaling

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U2 - 10.3390/cancers14102508

DO - 10.3390/cancers14102508

M3 - Article

C2 - 35626109

SN - 2072-6694

VL - 14

JO - Cancers

JF - Cancers

IS - 10

M1 - 2508

ER -

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

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