To preserve their integrity and function, cells must maintain tight control of their borders. One carefully regulated import mechanism, endocytosis, relies on physical deformation and invagination of the cell membrane to engulf extracellular components and form intracellular vesicles. Endocytosis has been predominantly studied in association with the protein clathrin, which coats the membrane and functions with adaptor proteins to direct incoming vesicular traffic to appropriate intracellular destinations. While studying one of the molecular components of membrane trafficking, the GTP-binding protein ARF6, Dr. Donaldson discovered that cells also operate a distinct endocytic pathway independent of clathrin. Since then, her laboratory has found that clathrin-independent endocytosis (CIE) occurs in every human cell type they have examined.
Dr. Donaldson's laboratory is focused on defining CIE: Is it a single system or are there multiple related pathways? What cargo proteins move through it? How is their internalization regulated? What is the impact on cellular function and organismal physiology?
Dr. Donaldson is also broadly interested in how proteins in the plasma membrane are degraded and replaced as well as in the relationship between protein turnover and different endocytic pathways. She and her colleagues use biochemical and genetic techniques to study the impact of mutations and analyze membrane trafficking through cellular imaging in live and fixed cells.
Dr. Donaldson and her colleagues first identified the ubiquitous MHC Class I molecules as a cargo for CIE. Since then, she has identified a number of nutrient transporters (e.g. for amino acids and glucose) as CIE cargo as well as proteins involved in intercellular interactions and cell-matrix interactions (e.g. CD44 and integrin proteins). In addition, many microorganisms, including certain bacteria and viruses, are able to enter cells by exploiting CIE mechanisms. CIE appears to operate as a quality control mechanism for the cell. Entry per se does not appear to be selective; however, once cargo is brought into the cell, the cell seems to have mechanisms to monitor the cargo in endosomes for sorting signals, misfolding, or other threats, and the ability to recycle cargo back out of the cell. From her earlier work with ARF6, Dr. Donaldson believes that CIE—and the recycling process in particular—is important for initiating specific forms of cytoskeletal movement. Dr. Donaldson's laboratory is identifying the sorting sequences on proteins that are processed through CIE and studying the cellular machinery that recognizes them.
Still in the early stages of uncovering the molecular machinery underlying CIE, Dr. Donaldson and her colleagues do not yet have selective tools to manipulate CIE. They are exploring FDA-approved compounds to discover pharmacological agents that either stimulate or inhibit CIE. Such an agent would certainly serve as a valuable experimental tool and could have therapeutic value.