RESUMEN
Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.
Asunto(s)
Membrana Celular/metabolismo , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Humanos , Integrinas/genética , Proteínas de la Membrana/genética , Ratones Noqueados , Movimiento (Física) , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación , Proteínas de Neoplasias/genética , Unión ProteicaRESUMEN
Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.
Asunto(s)
Movimiento Celular , Integrinas/metabolismo , Microscopía/métodos , Optogenética/métodos , Mapeo de Interacción de Proteínas/métodos , Imagen Individual de Molécula/métodos , Actinas/genética , Actinas/metabolismo , Animales , Adhesión Celular , Línea Celular Transformada , Criptocromos/genética , Criptocromos/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Expresión Génica , Integrinas/genética , Ratones , Microscopía/instrumentación , Miosinas/genética , Miosinas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Unión Proteica , Seudópodos/metabolismo , Seudópodos/ultraestructura , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cells are mechanical living machines that remodel their microenvironment by adhering and generating forces on the extracellular matrix (ECM) using integrin-dependent adhesion sites (IAS). In return, the biochemical and physical nature of the ECM determines cellular behavior and morphology during proliferation, differentiation and migration. IAS come in different shapes and forms. They have specific compositions, morphologies, mechanical and biochemical signaling activities, which serve different cellular functions. Proteomic studies showed that IAS are composed of a large repertoire of proteins that could be linked to different functional activities, including signaling, force-transmission and force-sensing. Thanks to recent technological advances in microscopy and protein engineering, it is now possible to localize single proteins in three dimensions inside IAS, determine their diffusive behaviors, orientations, and how much mechanical force is transmitted across individual components. Here, we review how researchers have used those tools to investigate how IAS components assemble and dynamically interact to produce diverse functions of adhesive structures.
Asunto(s)
Adhesión Celular/fisiología , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Sustancias Macromoleculares/metabolismo , Animales , Humanos , Mecanotransducción Celular/fisiología , Estrés MecánicoRESUMEN
To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.