Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

TLDR

Although the importance of clathrin‑ and caveolin‑independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural and electron tomographic analysis, the authors show that clathrin‑independent carriers (CLICs) comprise roughly three times the volume internalized by clathrin‑mediated endocytosis and are multidomain organelles that form a complex sorting station during maturation. Proteomic and functional analyses reveal that CLICs link adhesion turnover to migration, with polarized endocytosis of CD44 and Thy‑1 at the leading edge, and that transient CLIC ablation impairs fibroblast migration, establishing CLICs as the primary high‑capacity pathway for membrane turnover during cell movement.

Abstract

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.