The clathrin-independent endosomal system is required for cellular homeostasis and specialized modifications of the plasma membrane such as cell spreading and polarization. Clathrin-independent endocytosis (CIE) has been demonstrated in adherent cells including fibroblasts and epithelial cells. However, non-adherent cells also have highly dynamic clathrin-independent pathways, which have not been well described. Here, I have characterized Arf6-associated clathrin-independent endocytosis (CIE) in the human T cell line Jurkat and identified it's importance in immunological synapse formation. Our findings indicate that the CIE pathway is similar in Jurkat cells as compared to other cell types including rates of endocytosis and sorting after internalization. Two GTPases, Arf6 and Rab22, have been shown to regulate the clathrin-independent endosomal system and play a role in cell spreading. We found that wild type and constitutively active Arf6 co-localized with CIE cargo in resting T cells. Arf6 constitutively active mutant inhibited CIE cargo internalization but not internalization of CME cargo. Rab22 co-localized with CIE cargo at the endocytic-recycling compartment. Expression of the dominant negative Rab22 mutant also inhibited internalization of MHCI indicating it plays a direct role in CIE cargo internalization. T cells must modify their membranes to specifically interact with antigen presenting cells. To establish the role of CIE in this process, we then examined the role of Arf6 and Rab22 in T cell/antigen presenting cell conjugate formation. Both expression of dominant negative or constitutively active mutants of Arf6 reduced T cell conjugate formation while expression of only the Rab22 dominant negative mutant inhibited T cell/APC conjugate formation. Furthermore, T cells expressing the dominant negative mutant of Rab22 were not able to spread on antibody-coated coverslips that normally cause T cell activation. These results indicate that the clathrin independent endosomal system is required for membrane remodeling events necessary for T cell conjugate formation and T cell spreading during activation. I also conducted a proteomics screen to identify binding partners of CIE cargo proteins. I identified multiple proteins that could possibly play a role in CIE internalization and discovered a subset of proteins that specifically interact with A cargo proteins and not B cargo proteins. It is possible they could play a role in cargo retention at the plasma membrane or sorting after internalization. Three proteins of interest that interact with A cargo include NHERF-1 and ezrin, which participate in actin arrangements, and Dlg-1, a known scaffolding protein for synaptic vesicles. Ezrin and Dlg-1 co-localize with the CIE cargo protein CD98 in HeLa cells indicating that they could be interacting in cells.

The DEAD-Box RNA helicase family is a conserved group of enzymes that function in gene expression through ATP-dependent RNA unwinding and ribonucleoprotein (RNP) remodeling. DEAD-Box helicases function in multiple cellular processes, including pre-mRNA processing, translation, mRNA export, and mRNA decay. Although DEAD-Box proteins are critical for gene expression, much of their mechanistic activities are poorly understood. DEAD-Box proteins have increasingly been linked to tumorigenesis in humans, and better defining their activity at the mechanistic level will aid in understanding the underlying disease pathology. In this study, we used the model organism Saccharomyces cerevisiae to study the human DEAD-Box protein DDX3 orthologue, Ded1, and its role in translation initiation during cellular stress. Recently, we have found that Ded1 is an important mediator of the cellular stress response in a TOR-dependent manner. TOR regulates protein synthesis dependent on energy availability in the cell by regulating the assembly of the eukaryotic translation initiation complex. Human DDX3 has been found to interact with translation initiation complex subunit eIF4E and Ded1 has been found to interact with the translation initiation complex subunit eIF4G. In this study, we examined the purported interaction region between Ded1 and eIF4G on the C-terminus of Ded1 and found that ded1 Δ591-604 prevents eIF4G degradation under rapamycin treatment and confers resistance to rapamycin-induced growth inhibition. We also examined putative regulatory phosphorylation sites in the purported Ded1 eIF4G binding region. We propose that the Ded1/eIF4G interaction is critical for the repression of translation by Ded1 and that eIF4G degradation may be regulated by Ded1 under stress conditions.