RESUMEN
Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.
Asunto(s)
Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Mapeo de Interacción de Proteínas , Animales , Antígenos CD4/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Membrana Celular/química , Células Cultivadas , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Mapeo de Interacción de Proteínas/instrumentación , Mapeo de Interacción de Proteínas/métodos , Propiedades de SuperficieRESUMEN
We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.