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dc.contributor.advisorWilson, Jean M.en
dc.contributor.advisorDonaldson, Julie G.en
dc.contributor.authorJohnson, Debra L.
dc.creatorJohnson, Debra L.en
dc.date.accessioned2016-06-09T17:08:49Z
dc.date.available2016-06-09T17:08:49Z
dc.date.issued2016
dc.identifier.urihttp://hdl.handle.net/10150/612373
dc.description.abstractThe 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.
dc.language.isoen_USen
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en
dc.subjectClathrin-independenten
dc.subjectendocytosisen
dc.subjectmembrane traffickingen
dc.subjectRab22en
dc.subjectT cellsen
dc.subjectCellular and Molecular Medicineen
dc.subjectArf6en
dc.titleEngagement of T cells with Antigen Presenting Cells is Dependent on Clathrin-Independent Endocytic Trafficking: The Role of Arf6 and Rab22en_US
dc.typetexten
dc.typeElectronic Dissertationen
thesis.degree.grantorUniversity of Arizonaen
thesis.degree.leveldoctoralen
dc.contributor.committeememberLybarger, Lonnieen
dc.contributor.committeememberKuhns, Michael S.en
dc.contributor.committeememberWilson, Jean M.en
dc.contributor.committeememberDonaldson, Julie G.en
dc.description.releaseRelease after 13-May-2017en
thesis.degree.disciplineGraduate Collegeen
thesis.degree.disciplineCellular and Molecular Medicineen
thesis.degree.namePh.D.en
refterms.dateFOA2017-05-13T00:00:00Z
html.description.abstractThe 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.


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