@article {115047, title = {Reconstitution of Membrane-tethered Minimal Actin Cortices on Supported Lipid Bilayers.}, journal = {J Vis Exp}, year = {2022}, month = {2022 Jul 12}, 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.

}, keywords = {Actin Cytoskeleton, Actins, Cell Membrane, Lipid Bilayers, Myosins}, issn = {1940-087X}, doi = {10.3791/63968}, author = {K{\"o}ster, Darius Vasco and Bhat, Abrar and Talluri, Sankarshan and Mayor, Satyajit} } @article {62769, title = {A composition-dependent molecular clutch between T cell signaling condensates and actin.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Jul 03}, abstract = {

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS.

}, issn = {2050-084X}, doi = {10.7554/eLife.42695}, author = {Ditlev, Jonathon A and Vega, Anthony R and K{\"o}ster, Darius Vasco and Su, Xiaolei and Tani, Tomomi and Lakoduk, Ashley M and Vale, Ronald D and Mayor, Satyajit and Jaqaman, Khuloud and Rosen, Michael K} } @article {3546, title = {Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer.}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 Mar 22}, pages = {E1645-54}, abstract = {

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

}, issn = {1091-6490}, doi = {10.1073/pnas.1514030113}, author = {K{\"o}ster, Darius Vasco and Husain, Kabir and Iljazi, Elda and Bhat, Abrar and Bieling, Peter and Mullins, R Dyche and Rao, Madan and Mayor, Satyajit} }