Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
| Autor: | Anthony W. McCarthy, Gregor Reither, Alexander M. Walter, Martin Lehmann, Bassam Tawfik, Keimpe D. Wierda, Rainer Müller, Volker Haucke, Paulo S. Pinheiro, Martin Kruse, Bertil Hille, Carsten Schultz, Jens Rettig, Jakob B. Sørensen, André Nadler, Iwona Ziomkiewicz |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Phosphatidylinositol 4 5-Diphosphate optical uncaging Vesicle fusion phosphatidylinositols Mouse QH301-705.5 Science Chromaffin Cells Cytological Techniques Munc13 Nerve Tissue Proteins General Biochemistry Genetics and Molecular Biology Exocytosis Synaptotagmin 1 Cell Line 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Journal Article Ultraviolet light Animals Phosphatidylinositol Biology (General) adrenal chromaffin cell General Immunology and Microbiology General Neuroscience Vesicle Intracellular Signaling Peptides and Proteins Membrane Proteins General Medicine Cell Biology Cell biology synaptotagmin 030104 developmental biology Membrane protein chemistry Phosphatidylinositol 4 5-bisphosphate Synaptotagmin I Medicine Carrier Proteins mouse cell biology neuroscience exocytosis 030217 neurology & neurosurgery Research Article Neuroscience |
| Zdroj: | eLife eLife, 6:e30203 eLife, Vol 6 (2017) Walter, A M, Müller, R, Tawfik, B, Wierda, K D, Pinheiro, P S, Nadler, A, McCarthy, A W, Ziomkiewicz, I, Kruse, M, Reither, G, Rettig, J, Lehmann, M, Haucke, V, Hille, B, Schultz, C & Sorensen, J B 2017, ' Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis ', eLife, vol. 6, e30203 . https://doi.org/10.7554/eLife.30203 |
| ISSN: | 2050-084X |
| Popis: | 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. eLife digest 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. |
| Databáze: | OpenAIRE |
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