RESUMO
Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de srcRESUMO
In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here we present an assay that can detect curvature preference and membrane remodelling using free-floating liposomes using protein concentrations in a physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high curvature preference for the PH domain of AKT and the FYVE domain of HRS. In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of Epsin1 and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of Endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.