Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development

Clathrin-mediated endocytosis, which depends on the AP2 complex, plays an essential role in many cellular and developmental processes in mammalian cells. However, the function of the AP2 complex in plants remains largely unexplored. Here, we show in Arabidopsis that the AP2 sigma subunit mutant (ap2 sigma) displays various developmental defects that are similar to those of mutants defective in auxin transport and/or signaling, including single, trumpet-shaped and triple cotyledons, impaired vascular pattern, reduced vegetative growth, defective silique development and drastically reduced fertility. We demonstrate that AP2 sigma is closely associated and physically interacts with the clathrin light chain (CLC) in vivo using fluorescence cross-correlation spectroscopy (FCCS), protein proximity analyses and co-immunoprecipitation assays. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), we show that AP2 sigma-mCherry spots colocalize with CLC-EGFP at the plasma membrane, and that AP2 s-mCherry fluorescence appears and disappears before CLC-EGFP fluorescence. The density and turnover rate of the CLC-EGFP spots are significantly reduced in the ap2 sigma mutant. The internalization and recycling of the endocytic tracer FM4-64 and the auxin efflux carrier protein PIN1 are also significantly reduced in the ap2 sigma mutant. Further, the polar localization of PIN1-GFP is significantly disrupted during embryogenesis in the ap2 sigma mutant. Taken together, our results support an essential role of AP2 s in the assembly of a functional AP2 complex in plants, which is required for clathrin-mediated endocytosis, polar auxin transport and plant growth regulation.