RESUMO
Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Sinapses/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Clatrina/farmacologia , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HeLa , HumanosRESUMO
The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal-attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.
Assuntos
Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Edição de Genes , Proteínas de Fluorescência Verde/metabolismo , HumanosRESUMO
We characterized the tension response of clathrin-mediated endocytosis by using various cell manipulation methodologies. Elevated tension in a cell hinders clathrin-mediated endocytosis through inhibition of de novo coat initiation, elongation of clathrin coat lifetimes and reduction of high-magnitude growth rates. Actin machinery supplies an inward pulling force necessary for internalization of clathrin coats under high tension. These findings suggest that the physical cues cells receive from their microenvironment are major determinants of clathrin-mediated endocytic activity.
Assuntos
Actinas/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Células Epiteliais/metabolismo , Mecanotransdução Celular , Actinas/genética , Animais , Fenômenos Biomecânicos , Linhagem Celular , Linhagem Celular Tumoral , Tamanho Celular , Chlorocebus aethiops , Clatrina/genética , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Células Epiteliais/ultraestrutura , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Pressão Osmótica , Estresse MecânicoRESUMO
Current understanding of clathrin-mediated endocytosis (CME) dynamics is based on detection and tracking of fluorescently tagged clathrin coat components within cultured cells. Because of technical limitations inherent to detection and tracking of single fluorescent particles, CME dynamics is not characterized in vivo, so the effects of mechanical cues generated during development of multicellular organisms on formation and dissolution of clathrin-coated structures (CCSs) have not been directly observed. Here, we use growth rates of fluorescence signals obtained from short CCS intensity trace fragments to assess CME dynamics. This methodology does not rely on determining the complete lifespan of individual endocytic assemblies. Therefore, it allows for real-time monitoring of spatiotemporal changes in CME dynamics and is less prone to errors associated with particle detection and tracking. We validate the applicability of this approach to in vivo systems by demonstrating the reduction of CME dynamics during dorsal closure of Drosophila melanogaster embryos.