Date of Award
The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. Clathrin functions with a network of interacting accessory proteins, one of which is the adaptor complex AP-2, to co-ordinate vesicle formation. Disruption of genes involved in clathrin-mediated endocytosis causes embryonic lethality in multicellular animals suggesting that clathrin-mediated endocytosis is a fundamental cellular process. However, loss of clathrin-mediated endocytosis genes in single cell eukaryotes, such as S.cerevisiae (yeast), does not cause lethality, suggesting that clathrin may convey specific advantages for multicellularity. Furthermore, the spatiotemporal dynamics and requirements for individual components of the clathrin-mediated endocytic pathway differ between yeast and mammals. I therefore sought to study the components of clathrin-mediated endocytosis in another unicellular system, the organism Dictyostelium, which diverged early from the lineage leading to yeast and mammals. Dictyostelium offers a unique advantage as upon starvation it transitions from a unicellular to multicellular state. In this thesis I studied clathrin-mediated endocytosis in the unicellular growth phase of Dictyostelium and identified a heterotetrameric AP-2 complex in Dictyostelium that is homologous to that present in mammalian cells. Analysis of this pathway at a high spatial and temporal resolution shows the high degree of similarity in the kinetics of internalization of individual clathrin-coated vesicles between Dictyostelium and mammalian cells. These similarities support the conclusion that the formation of clathrin-coated vesicles is homologous between Dictyostelium and mammals and, thus, these features likely evolved early. I also found a role for clathrin-mediated endocytosis in maintenance and biogenesis of the contractile vacuole in Dictyostelium. Contractile vacuoles are specialized organelles found in some single celled organisms that allow cells to osmoregulate by collecting and removing excess water from the cytoplasm; they are not found in yeast or animals. I found that that the contractile vacuole protein, dajumin-GFP, is trafficked via the cell membrane and is a cargo that is internalized by clathrin-mediated endocytosis in Dictyostelium. Internalization of dajumin-GFP is via a clathrin-dependent, AP-2 independent mechanism and is distinct from other endocytic mechanisms. These results suggest the role of clathrin in protein sorting also evolved early, while dependence on specific components of the clathrin-mediated endocytic pathway, may have evolved later, as demonstrated by internalization independent of AP-2 function. In Dictyostelium, clathrin is known to be required for the formation of multicellular structures. Since it is possible to visualize the transition from a unicellular state to multicellularity, Dictyostelium is now uniquely positioned to study the dynamics of clathrin-mediated endocytosis during multicellular development.
Macro, Laura, "Conserved and Novel Properties of Clathrin-Mediated Endocytosis in Dictyostelium Discoideum" (2012). Student Theses and Dissertations. 163.