RESUMO
Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.
Assuntos
Actinas/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Fusão de Membrana/fisiologia , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos/genética , Animais , Evolução Biológica , Fusão Celular/métodos , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Evolução Molecular , Humanos , Orthoreovirus/genética , Ligação Proteica/genética , Reoviridae/genética , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteínas não Estruturais Virais/metabolismo , Internalização do VírusRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/ultraestrutura , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/genética , Polaridade Celular , Quelantes , Galinhas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Técnicas In Vitro , Bicamadas Lipídicas , Proteínas Luminescentes/genética , Microscopia Eletrônica , Modelos Biológicos , Níquel , Ácido Nitrilotriacético/análogos & derivados , Fosfatidilcolinas , Fosfatidiletanolaminas , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Propriedades de SuperfícieRESUMO
The cell membrane, the boundary that separates living cells from their environment, has been the subject of study for over a century. The fluid-mosaic model of Singer and Nicolson in 1972 proposed the plasma membrane as a two-dimensional fluid composed of lipids and proteins. Fifty years hence, advances in biophysical and biochemical tools, particularly optical imaging techniques, have allowed for a better understanding of the physical nature, organization, and composition of cell membranes. This has been made possible by visualizing membrane heterogeneities and their dynamics and appreciating the asymmetrical arrangement of lipids in living cell membranes. Despite these advances, mechanisms underlying the local spatiotemporal organization of membrane components remain unclear. This review surveys various models of membrane organization, culminating in a new model that incorporates nonequilibrium processes and forces exerted by interactions with extramembrane elements such as the actin cytoskeleton. The proposed model provides a comprehensive understanding of membrane organization, taking into account the dynamic nature of the cell membrane and its interactions with its immediate environment.
Assuntos
Lipídeos de Membrana , Proteínas , Lipídeos de Membrana/análise , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membrana Celular/metabolismo , Proteínas/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
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.
Assuntos
Actinas , Bicamadas Lipídicas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Miosinas/metabolismoRESUMO
Recent in vivo studies reveal that several membrane proteins are driven to form nanoclusters by active contractile flows arising from localized dynamic patterning of F-actin and myosin at the cortex. Since myosin-II assemble as minifilaments with tens of myosin heads, one might worry that steric considerations would obstruct the emergence of nanoclustering. Using coarse-grained, agent-based simulations that account for steric constraints, we find that the patterns exhibited by actomyosin in two dimensions, do not resemble the steady-state patterns in our in vitro reconstitution of actomyosin on a supported bilayer. We perform simulations in a thin rectangular slab, separating the layer of actin filaments from myosin-II minifilaments. This recapitulates the observed features of in vitro patterning. Using super resolution microscopy, we find evidence for such stratification in our in vitro system. Our study suggests that molecular stratification may be an important organizing feature of the cortical cytoskeleton in vivo.