Live-cell imaging of early coat protein dynamics during clathrin-mediated endocytosis

Clathrin-mediated endocytosis is an essential process that is mediated by the stepwise appearance or disappearance of many different proteins at the plasma membrane. In the budding yeast, these proteins are categorized into at least five modules, according to their spatiotemporal dynamics. Among them, the dynamics of proteins in the late coat module are well characterized, but those in the early coat module still remain unclear because of the lack of a suitable fluorescent marker with sufficient brightness to allow analysis. To examine the dynamics of early coat proteins, in this study we tagged four representative early coat proteins with 3GFP, and expressed them in a single cell. This cell exhibited a significant increase in the fluorescence intensity of early coat proteins relative to that of each 3GFP-tagged protein. Using this strain, we performed a detailed analysis of early coat proteins, including their precise lifetime, changes in fluorescence intensity, and motility on the plasma membrane. We found that early coat proteins move on the plasma membrane before internalization. Additionally, we expressed these 3GFP-tagged proteins in mutants with deletion of genes related to endocytosis, and found four mutants - end3Δ, las17Δ, sla2Δ, and clc1Δ- in which the lifetime of early coat proteins was markedly increased. Interestingly, deletion of the CLC1 gene dramatically reduced the internalization of early coat proteins whereas internalization of actin patches was largely unchanged, suggesting that the clc1Δ mutant might have a defect in the link between the early coat and actin modules.