Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(...

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Vydáno v:eLife Ročník 6
Hlavní autoři: Walter, Alexander M, Müller, Rainer, Tawfik, Bassam, Wierda, Keimpe DB, Pinheiro, Paulo S, Nadler, André, McCarthy, Anthony W, Ziomkiewicz, Iwona, Kruse, Martin, Reither, Gregor, Rettig, Jens, Lehmann, Martin, Haucke, Volker, Hille, Bertil, Schultz, Carsten, Sørensen, Jakob Balslev
Médium: Journal Article
Jazyk:angličtina
Vydáno: England eLife Sciences Publications Ltd 25.10.2017
eLife Sciences Publications, Ltd
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ISSN:2050-084X, 2050-084X
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Abstract Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors. Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next. Packages called vesicles store transmitters within the signaling cell. When the cell needs to send a signal, the vesicles fuse with the cell's membrane and release their cargo. For many signaling processes, such as those used by neurons, this fusion is regulated, fast, and coupled to the signal that the cell receives to activate release. Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen. One signaling lipid called PI(4,5)P2 (short for phosphatidylinositol 4,5-bisphosphate) is essential for vesicle fusion as well as for other processes in cells. It interacts with several proteins that help it control fusion and the release of transmitter. While it is possible to study the role of these proteins using genetic tools to inactivate them, the signaling lipids are more difficult to manipulate. Existing methods result in slow changes in PI(4,5)P2 levels, making it hard to directly attribute later changes to PI(4,5)P2. Walter, Müller, Tawfik et al. developed a new method to measure how PI(4,5)P2 affects transmitter release in living mammalian cells, which causes a rapid increase in PI(4,5)P2 levels. The method uses a chemical compound called “caged PI(4,5)P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it. The ultraviolet light uncages the compound, generating active PI(4,5)P2 in less than one second. Walter et al. found that when they uncaged PI(4,5)P2 in this way, the amount of transmitter released by cells increased. Combining this with genetic tools, it was possible to investigate which proteins of the release machinery were required for this effect. The results suggest that two different types of proteins that interact with PI(4,5)P2 are needed: one must bind PI(4,5)P2 to carry out its role and the other helps PI(4,5)P2 accumulate at the site of vesicle fusion. The new method also allowed Walter et al. to show that a fast increase in PI(4,5)P2 triggers a subset of vesicles to fuse very rapidly. This shows that PI(4,5)P2 rapidly regulates the release of transmitter. Caged PI(4,5)P2 will be useful to study other processes in cells that need PI(4,5)P2, helping scientists understand more about how signaling lipids control many different events at cellular membranes.
AbstractList Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors. Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next. Packages called vesicles store transmitters within the signaling cell. When the cell needs to send a signal, the vesicles fuse with the cell's membrane and release their cargo. For many signaling processes, such as those used by neurons, this fusion is regulated, fast, and coupled to the signal that the cell receives to activate release. Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen. One signaling lipid called PI(4,5)P2 (short for phosphatidylinositol 4,5-bisphosphate) is essential for vesicle fusion as well as for other processes in cells. It interacts with several proteins that help it control fusion and the release of transmitter. While it is possible to study the role of these proteins using genetic tools to inactivate them, the signaling lipids are more difficult to manipulate. Existing methods result in slow changes in PI(4,5)P2 levels, making it hard to directly attribute later changes to PI(4,5)P2. Walter, Müller, Tawfik et al. developed a new method to measure how PI(4,5)P2 affects transmitter release in living mammalian cells, which causes a rapid increase in PI(4,5)P2 levels. The method uses a chemical compound called “caged PI(4,5)P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it. The ultraviolet light uncages the compound, generating active PI(4,5)P2 in less than one second. Walter et al. found that when they uncaged PI(4,5)P2 in this way, the amount of transmitter released by cells increased. Combining this with genetic tools, it was possible to investigate which proteins of the release machinery were required for this effect. The results suggest that two different types of proteins that interact with PI(4,5)P2 are needed: one must bind PI(4,5)P2 to carry out its role and the other helps PI(4,5)P2 accumulate at the site of vesicle fusion. The new method also allowed Walter et al. to show that a fast increase in PI(4,5)P2 triggers a subset of vesicles to fuse very rapidly. This shows that PI(4,5)P2 rapidly regulates the release of transmitter. Caged PI(4,5)P2 will be useful to study other processes in cells that need PI(4,5)P2, helping scientists understand more about how signaling lipids control many different events at cellular membranes.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors. Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next. Packages called vesicles store transmitters within the signaling cell. When the cell needs to send a signal, the vesicles fuse with the cell's membrane and release their cargo. For many signaling processes, such as those used by neurons, this fusion is regulated, fast, and coupled to the signal that the cell receives to activate release. Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen. One signaling lipid called PI(4,5)P2 (short for phosphatidylinositol 4,5-bisphosphate) is essential for vesicle fusion as well as for other processes in cells. It interacts with several proteins that help it control fusion and the release of transmitter. While it is possible to study the role of these proteins using genetic tools to inactivate them, the signaling lipids are more difficult to manipulate. Existing methods result in slow changes in PI(4,5)P2 levels, making it hard to directly attribute later changes to PI(4,5)P2. Walter, Müller, Tawfik et al. developed a new method to measure how PI(4,5)P2 affects transmitter release in living mammalian cells, which causes a rapid increase in PI(4,5)P2 levels. The method uses a chemical compound called “caged PI(4,5)P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it. The ultraviolet light uncages the compound, generating active PI(4,5)P2 in less than one second. Walter et al. found that when they uncaged PI(4,5)P2 in this way, the amount of transmitter released by cells increased. Combining this with genetic tools, it was possible to investigate which proteins of the release machinery were required for this effect. The results suggest that two different types of proteins that interact with PI(4,5)P2 are needed: one must bind PI(4,5)P2 to carry out its role and the other helps PI(4,5)P2 accumulate at the site of vesicle fusion. The new method also allowed Walter et al. to show that a fast increase in PI(4,5)P2 triggers a subset of vesicles to fuse very rapidly. This shows that PI(4,5)P2 rapidly regulates the release of transmitter. Caged PI(4,5)P2 will be useful to study other processes in cells that need PI(4,5)P2, helping scientists understand more about how signaling lipids control many different events at cellular membranes.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P ] is essential for exocytosis. Classical ways of manipulating PI(4,5)P levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P , which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P activation of exocytosis did not depend on the PI(4,5)P -binding CAPS-proteins, suggesting that PI(4,5)P uncaging may bypass CAPS-function. Finally, PI(4,5)P uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
Author Kruse, Martin
Reither, Gregor
Rettig, Jens
Hille, Bertil
Müller, Rainer
Wierda, Keimpe DB
Sørensen, Jakob Balslev
Nadler, André
Schultz, Carsten
McCarthy, Anthony W
Walter, Alexander M
Ziomkiewicz, Iwona
Tawfik, Bassam
Lehmann, Martin
Haucke, Volker
Pinheiro, Paulo S
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  surname: Walter
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  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
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  givenname: Rainer
  orcidid: 0000-0003-3464-494X
  surname: Müller
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  organization: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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  givenname: Bassam
  orcidid: 0000-0003-1193-8494
  surname: Tawfik
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  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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  givenname: Keimpe DB
  orcidid: 0000-0002-8784-9490
  surname: Wierda
  fullname: Wierda, Keimpe DB
  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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  givenname: Paulo S
  surname: Pinheiro
  fullname: Pinheiro, Paulo S
  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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  givenname: André
  surname: Nadler
  fullname: Nadler, André
  organization: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
– sequence: 7
  givenname: Anthony W
  orcidid: 0000-0002-3771-351X
  surname: McCarthy
  fullname: McCarthy, Anthony W
  organization: Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
– sequence: 8
  givenname: Iwona
  surname: Ziomkiewicz
  fullname: Ziomkiewicz, Iwona
  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, Discovery Sciences, AstraZeneca, Cambridge, United Kingdom
– sequence: 9
  givenname: Martin
  surname: Kruse
  fullname: Kruse, Martin
  organization: Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, United States
– sequence: 10
  givenname: Gregor
  surname: Reither
  fullname: Reither, Gregor
  organization: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
– sequence: 11
  givenname: Jens
  surname: Rettig
  fullname: Rettig, Jens
  organization: Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
– sequence: 12
  givenname: Martin
  surname: Lehmann
  fullname: Lehmann, Martin
  organization: Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
– sequence: 13
  givenname: Volker
  surname: Haucke
  fullname: Haucke, Volker
  organization: Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
– sequence: 14
  givenname: Bertil
  surname: Hille
  fullname: Hille, Bertil
  organization: Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, United States
– sequence: 15
  givenname: Carsten
  surname: Schultz
  fullname: Schultz, Carsten
  organization: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
– sequence: 16
  givenname: Jakob Balslev
  orcidid: 0000-0001-5465-3769
  surname: Sørensen
  fullname: Sørensen, Jakob Balslev
  organization: Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
BackLink https://www.ncbi.nlm.nih.gov/pubmed/29068313$$D View this record in MEDLINE/PubMed
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2017, Walter et al 2017 Walter et al
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Keywords mouse
adrenal chromaffin cell
cell biology
Munc13
neuroscience
optical uncaging
exocytosis
synaptotagmin
phosphatidylinositols
Language English
License http://creativecommons.org/licenses/by/4.0
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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These authors contributed equally to this work.
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Snippet Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism,...
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P ] is essential for exocytosis. Classical ways of manipulating PI(4,5)P levels are slower than its metabolism,...
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SubjectTerms adrenal chromaffin cell
Adrenal glands
Animals
Biology
Biophysics
Calcium
Carrier Proteins - metabolism
Cell Biology
Cell Line
Chromaffin cells
Chromaffin Cells - metabolism
Cytological Techniques - methods
Enzymes
Exocytosis
Experiments
Intracellular Signaling Peptides and Proteins - metabolism
Kinases
Lipids
Membrane Proteins - metabolism
Metabolism
Metabolites
Mice
Munc13
Nerve Tissue Proteins - metabolism
Neuroscience
optical uncaging
Phosphatidylinositol 4,5-diphosphate
Phosphatidylinositol 4,5-Diphosphate - metabolism
phosphatidylinositols
Physiology
Plasma
Proteins
Signal transduction
Synaptotagmin
Synaptotagmin I - metabolism
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Title Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
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