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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 ISSN 1940-087X.

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Official URL: http://dx.doi.org/10.3791/63968

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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
Subjects: Q Science > QH Natural history
Q Science > QP Physiology
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
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:
DateEvent
12 July 2022Published
13 July 2022Accepted
Number: 185
Article Number: e63968
DOI: 10.3791/63968
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access (Creative Commons)
Date of first compliant deposit: 20 July 2022
Date of first compliant Open Access: 20 July 2022
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
UNSPECIFIEDAXA Research Fundhttp://dx.doi.org/10.13039/501100001961
Warwick-Wellcome Quantitative Biomedicine Programme : Wellcome ISSF, RMRCB0058University of Warwickhttp://dx.doi.org/10.13039/501100000741
Warwick-Wellcome Quantitative Biomedicine Programme : Wellcome ISSF, RMRCB0058Wellcome Trusthttp://dx.doi.org/10.13039/100010269
IA/M/15/1/502018The Wellcome Trust DBT India Alliancehttp://dx.doi.org/10.13039/501100009053

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