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A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases

Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the...

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Autores principales: Mavrantoni, Angeliki, Thallmair, Veronika, Leitner, Michael G., Schreiber, Daniela N., Oliver, Dominik, Halaszovich, Christian R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379879/
https://www.ncbi.nlm.nih.gov/pubmed/25873899
http://dx.doi.org/10.3389/fphar.2015.00068
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author Mavrantoni, Angeliki
Thallmair, Veronika
Leitner, Michael G.
Schreiber, Daniela N.
Oliver, Dominik
Halaszovich, Christian R.
author_facet Mavrantoni, Angeliki
Thallmair, Veronika
Leitner, Michael G.
Schreiber, Daniela N.
Oliver, Dominik
Halaszovich, Christian R.
author_sort Mavrantoni, Angeliki
collection PubMed
description Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P(2) and PI(3,4,5)P(3) levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P(3). Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P(2) and PI(3,4,5)P(3) levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.
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spelling pubmed-43798792015-04-13 A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases Mavrantoni, Angeliki Thallmair, Veronika Leitner, Michael G. Schreiber, Daniela N. Oliver, Dominik Halaszovich, Christian R. Front Pharmacol Pharmacology Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P(2) and PI(3,4,5)P(3) levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P(3). Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P(2) and PI(3,4,5)P(3) levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells. Frontiers Media S.A. 2015-03-31 /pmc/articles/PMC4379879/ /pubmed/25873899 http://dx.doi.org/10.3389/fphar.2015.00068 Text en Copyright © 2015 Mavrantoni, Thallmair, Leitner, Schreiber, Oliver and Halaszovich. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Pharmacology
Mavrantoni, Angeliki
Thallmair, Veronika
Leitner, Michael G.
Schreiber, Daniela N.
Oliver, Dominik
Halaszovich, Christian R.
A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title_full A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title_fullStr A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title_full_unstemmed A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title_short A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases
title_sort method to control phosphoinositides and to analyze pten function in living cells using voltage sensitive phosphatases
topic Pharmacology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379879/
https://www.ncbi.nlm.nih.gov/pubmed/25873899
http://dx.doi.org/10.3389/fphar.2015.00068
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