Mosaic evolution of clathrin-mediated endocytosis in fungi

Clathrin-mediated endocytosis is an ancient eukaryotic trafficking pathway, which transports plasma membrane and associated cargo into the cell and is involved in numerous cell- and tissue-level processes. Cargo selection and clathrin-coated vesicle formation are mediated by over 60 proteins that assemble in a regular and sequential manner at the plasma membrane. Decades of endocytosis studies have followed the tenet that uncovering the conserved core molecular mechanisms is sufficient to understand a cellular process. However, this approach also revealed a number of cell-type- or species-related variations that challenge the notion of a universally conserved, core mechanism. In this paper, we refocus on the endocytic diversity to understand how evolution shapes endocytic mechanisms. We define a comparative evolutionary cell biology approach that uses dikarya fungi as a model clade and live-cell fluorescence microscopy to study endocytosis dynamics in three species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our results quantitatively define several phenotypic differences between the species. We uncover differences that impact the endocytic early phase, the protein assembly order, actin regulation, membrane invagination, and scission. These findings demonstrate a mosaic evolution of endocytic traits, suggesting ancestral states and directions of change. We also investigate phenotypic plasticity and robustness against environmental conditions. Lastly, we demonstrate that relatively minor evolutionary changes can majorly impact endocytic phenotypes. These findings promote an appreciation of endocytic variation as not auxiliary, but vital to the mechanistic understanding of this conserved cellular pathway.