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Reconstitution of membrane-tethered minimal actin cortices on supported lipid bilayers
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Köster, Darius Vasco, Bhat, Abrar, Talluri, Sankarshan and Mayor, Satyajit (2022) Reconstitution of membrane-tethered minimal actin cortices on supported lipid bilayers. Journal of Visualized Experiments (185). e63968. doi:10.3791/63968
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Official URL: http://dx.doi.org/10.3791/63968
Abstract
The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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| Library of Congress Subject Headings (LCSH): | Bilayer lipid membranes, Actin, Microfilament proteins | |||||||||||||||
| Journal or Publication Title: | Journal of Visualized Experiments | |||||||||||||||
| Publisher: | JoVE | |||||||||||||||
| ISSN: | 1940-087X | |||||||||||||||
| Official Date: | 12 July 2022 | |||||||||||||||
| Dates: |
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| Number: | 185 | |||||||||||||||
| Article Number: | e63968 | |||||||||||||||
| DOI: | 10.3791/63968 | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access | |||||||||||||||
| RIOXX Funder/Project Grant: |
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