Internalization of Non-Cx43 Gap Junctions

Abstract Endocytosis is critical for the maintenance of multicellular life. Cells need to be able to endocytose effector molecules from their neighbors to carry out tasks for growth and development, regulate immune responses, maintain metabolic pathways, etc. Remarkably some cells can not only endocytose their own membrane, but also pieces of an adjoining cell's membrane as well as part of its neighbor's cytoplasm through a process called trans-endocytosis. Gap junctions, the clusters of trans-membrane channels that provide cells with the capability of direct cell-to-cell communication, provide a great model to explore the dynamics of trans-endocytosis because of the means by which gap junction endocytosis takes place. In this thesis, (1) I give insight into the abilities of gap junctions, comprised of different members of the connexin protein family, to be endocytosed from one cell to another. I identify all canonical endocytic motifs throughout the connexin family as well as several possible non-canonical motifs. I demonstrate that some canonical endocytic motifs are dispensable for the endocytosis of several connexins in HeLa cells. (2) I also explore the endocytosis mechanism of Cx36 in greater detail and demonstrate that its endocytosis may be regulated by clathrin and Dynamin2. I further demonstrate that large pieces of Cx36 can be deleted without any inhibition to its endocytic ability. I show through cell staining that Cx36 interacts with ubiquitin but for an un-identified purpose. (4) Although the complete mechanism of Cx36, and connexin endocytosis as a whole, is not fully established in this thesis, I propose a variety of possible mechanisms as well as techniques that the field could utilize in the future to better understand this process.