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Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation

In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circum...

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Autores principales: Kuchler, Oliver, Gerlach, Jule, Vomhof, Thomas, Hettich, Johannes, Steinmetz, Julia, Gebhardt, J. Christof M., Michaelis, Jens, Knöll, Bernd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9090491/
https://www.ncbi.nlm.nih.gov/pubmed/35537478
http://dx.doi.org/10.1098/rsob.210383
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author Kuchler, Oliver
Gerlach, Jule
Vomhof, Thomas
Hettich, Johannes
Steinmetz, Julia
Gebhardt, J. Christof M.
Michaelis, Jens
Knöll, Bernd
author_facet Kuchler, Oliver
Gerlach, Jule
Vomhof, Thomas
Hettich, Johannes
Steinmetz, Julia
Gebhardt, J. Christof M.
Michaelis, Jens
Knöll, Bernd
author_sort Kuchler, Oliver
collection PubMed
description In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation.
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spelling pubmed-90904912022-05-11 Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation Kuchler, Oliver Gerlach, Jule Vomhof, Thomas Hettich, Johannes Steinmetz, Julia Gebhardt, J. Christof M. Michaelis, Jens Knöll, Bernd Open Biol Research In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation. The Royal Society 2022-05-11 /pmc/articles/PMC9090491/ /pubmed/35537478 http://dx.doi.org/10.1098/rsob.210383 Text en © 2022 The Authors. https://creativecommons.org/licenses/by/4.0/Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, provided the original author and source are credited.
spellingShingle Research
Kuchler, Oliver
Gerlach, Jule
Vomhof, Thomas
Hettich, Johannes
Steinmetz, Julia
Gebhardt, J. Christof M.
Michaelis, Jens
Knöll, Bernd
Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title_full Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title_fullStr Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title_full_unstemmed Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title_short Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
title_sort single-molecule tracking (smt) and localization of srf and mrtf transcription factors during neuronal stimulation and differentiation
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9090491/
https://www.ncbi.nlm.nih.gov/pubmed/35537478
http://dx.doi.org/10.1098/rsob.210383
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