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Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry

Boštjan Rituper, Alenka Guček, Marjeta Lisjak, Urszula Gorska, Aleksandra Šakanović, Saša Trkov Bobnar, Eva Lasič, Mićo Božić, Prabhodh S. Abbineni, Jernej Jorgačevski, Marko Kreft, Alexei Verkhratsky, Frances M. Platt, Gregor Anderluh, Matjaž Stenovec, Bojan Božič, Jens R. Coorssen, View ORCID ProfileRobert Zorec
doi: https://doi.org/10.1101/2020.04.06.027169
Boštjan Rituper
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
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Alenka Guček
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
10Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
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Marjeta Lisjak
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
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Urszula Gorska
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
11Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
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Aleksandra Šakanović
2Celica Biomedical, 1000, Ljubljana, Slovenia
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Saša Trkov Bobnar
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
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Eva Lasič
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
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Mićo Božić
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
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Prabhodh S. Abbineni
4Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-5632, USA
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Jernej Jorgačevski
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
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Marko Kreft
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
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Alexei Verkhratsky
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
5Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK
6Achucarro Center for Neuroscience, IKERBASQUE, 48011 Bilbao, Spain
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Frances M. Platt
7Department of Pharmacology, University of Oxford, Oxford, OX1 3QT, UK
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Gregor Anderluh
2Celica Biomedical, 1000, Ljubljana, Slovenia
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Matjaž Stenovec
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
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Bojan Božič
8Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia
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Jens R. Coorssen
9Department of Health Sciences, Faculty of Applied Health Sciences and Department of Biological Sciences, Faculty of Mathematics & Science, Brock University, St Catherine’s, Ontario, Canada
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Robert Zorec
1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
3Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
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  • ORCID record for Robert Zorec
  • For correspondence: robert.zorec@mf.uni-lj.si
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ABSTRACT

Eukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipids remains unclear. Super-resolution microscopy revealed that lactotroph secretory vesicles discharge cholesterol after stimulation of exocytosis; subsequently, vesicle cholesterol redistributes to the outer leaflet of the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes release of vesicle hormone, indicating that cholesterol constricts the fusion pore. A new model of cholesterol-dependent fusion pore diameter regulation is proposed. High-resolution measurements of fusion pore conductance confirmed that the fusion pore widens with cholesterol depletion and constricts with cholesterol enrichment. In fibroblasts lacking the Npc1 protein, in which cholesterol accumulates in vesicles, the fusion pore is narrower than in controls, showing that cholesterol regulates fusion pore geometry.

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Graphical AbstractTop: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the outer leaflet of the plasmalemma controls fusion pore constriction.

In Brief A membrane pore is formed when the vesicle membrane fuses with the plasmalemma. Proteins were considered key regulators of the opening and closing of this fusion pore. Here, evidence is provided to show that cholesterol, a membrane constituent, determines a radial force constricting the fusion pore, revealing that the fusion pore functions as a proteolipidic structure.

Highlights

  • Intravesicular cholesterol redistributes to the outer leaflet of the plasmalemma.

  • Cholesterol depletion widens the fusion pore, whereas cholesterol enrichment constricts the fusion pore.

  • A model of cholesterol-dependent force preventing fusion pore widening is developed.

  • Disease-related increase in vesicle cholesterol constricts the fusion pore.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 06, 2020.
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Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry
Boštjan Rituper, Alenka Guček, Marjeta Lisjak, Urszula Gorska, Aleksandra Šakanović, Saša Trkov Bobnar, Eva Lasič, Mićo Božić, Prabhodh S. Abbineni, Jernej Jorgačevski, Marko Kreft, Alexei Verkhratsky, Frances M. Platt, Gregor Anderluh, Matjaž Stenovec, Bojan Božič, Jens R. Coorssen, Robert Zorec
bioRxiv 2020.04.06.027169; doi: https://doi.org/10.1101/2020.04.06.027169
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Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry
Boštjan Rituper, Alenka Guček, Marjeta Lisjak, Urszula Gorska, Aleksandra Šakanović, Saša Trkov Bobnar, Eva Lasič, Mićo Božić, Prabhodh S. Abbineni, Jernej Jorgačevski, Marko Kreft, Alexei Verkhratsky, Frances M. Platt, Gregor Anderluh, Matjaž Stenovec, Bojan Božič, Jens R. Coorssen, Robert Zorec
bioRxiv 2020.04.06.027169; doi: https://doi.org/10.1101/2020.04.06.027169

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