Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

During inflammatory responses, macrophages make and secrete cytokines, including the proinflammatory cytokine TNF-alpha (TNF). TNF is a highly potent activator of immune responses with pleiomorphic effects throughout the body. TNF is a key causative agent of chronic inflammatory diseases and is of an intense clinical interest as a therapeutic target. At the outset of this thesis, little was known about how macrophages secrete TNF. Notably, the pathways, carriers and molecules that regulate TNF secretion had not been characterised. A main goal of this work was to identify compartment and molecules involved in the intracellular trafficking of TNF. Live cell imaging of GFP-TNF was established and this provided novel and important new insights into trafficking. Both endogenous and GFP-tagged TNF were followed in macrophages using fluorescence microscopy. The trafficking of other molecules in macrophages was also studied. The major findings of this work include the identification of a new two-step secretory pathway for TNF and other proteins from the trans-Golgi Network (TGN) to the cell surface. This pathway goes via the recycling endosome as an intermediate station. Pleiotrophic tubular-vesicular carriers containing TNF bud off the TGN for the post-Golgi trafficking of TNF and their characterization both in live cell imaging and in biochemical analysis of isolated vesicles constituted the main parts of this work. Functional studies, including endosome inactivation and overexpression of Rab11 mutants (proteins functioning at the level of the recycling endosome) revealed that recycling endosomes have indeed an essential role in the exocytic trafficking of TNF in macrophages. This thesis also provides further insight into recycling endosomes as a possible intermediate step in the exocytic trafficking of several other proteins including the adhesion protein E-Cadherin, that function at the cell surface. Finally, the last chapter of this thesis examines endocytic pathways in activated macrophages. Assays for fluid phase endocytosis and receptor-mediated endocytosis were established and the regulation of both pathways was compared. The results show that LPS has opposite effects on fluid phase and receptor mediated endocytosis, decreasing and increasing their activity respectively. Recycling of transferrin through the recycling endosome was also measured, providing a link with studies on TNF exocytosis. Overall, the work in this thesis has made a major contribution to our understanding of TNF trafficking in macrophages, of macrophage pathways more generally and of trafficking at a fundamental level. The findings herein set the stage for more in depth analysis at a single molecular level to explore TNF regulation in normal and disease cells.

Thesis (Ph.D.)--Case Western Reserve University, 2009 / Department of Biomedical Engineering Abstract Title from OhioLINK abstract screen (viewed on 10 April 2009) Available online via the OhioLINK ETD Center

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 150-161.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 19, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). "November 2006." Includes bibliographical references (p. [153]-161). Also available via the World Wide Web.

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La dissémination des cellules cancéreuses et la formation de métastases sont des étapes cruciales dans la progression tumorale et constituent une cause majeure des décès dus au cancer. La métalloprotéase transmembranaire MT1-MMP est un acteur clé impliqué dans le franchissement des barrières tissulaires et le remodelage de la matrice extracellulaire (ECM) par les cellules cancéreuses. MT1-MMP est présente dans des vésicules intracellulaires, appelées endosomes, via lesquels elle est adressée à la membrane plasmique (PM) afin d'y dégrader la ECM. Des travaux menés au laboratoire ont identifié le complexe exocyste (CE) comme un acteur important pour la formation d'invadopodes dans la lignée d'adénocarcinome mammaire MDA-MB-231. Ce complexe multiprotéique (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 et Exo84) est impliqué dans l'arrimage des vésicules intracellulaires à la PM. Des cribles double-hybride ont identifiés la protéine WASH comme partenaire potentiel du CE (via les sous-unités Exo84 et Sec3). WASH est capable d'induire la polymérisation de l'actine en activant le complexe Arp2/3. In vitro, nous avons montré que les complexes WASH et exocyste interagissent physiquement et coordonnent le trafic intracellulaire et l'adressage de MT1-MMP à la PM. Ces résultats mettent en évidence une étroite collaboration entre le cytosquelette d'actine et les mécanismes d'exocytose lors des étapes précoces de dégradation de la ECM ainsi que dans l'invasion tumorale. / Cancer cell invasion is a prerequisite to tumor progression and metastasis. In order to disseminate, tumor cells must degrade and remodel the extracellular matrix (ECM) in a process that requires the trans-membrane matrix metalloproteinase MT1-MMP, which is a key component of the ECM remodeling apparatus of cancer cells. MT1-MMP overexpression in cancers is associated with increased invasion and metastasis. Many cellular proteins are involved in the transport and delivery of MT1-MMP-containing vesicles to the PM. Previous work from the laboratory identified the exocyst complex (EC) as a key component required for matrix proteolysis and invasion of cancer cells. This multiprotein complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) plays essential roles in docking secretory vesicles at the PM for exocytosis. To better characterize this complex, a yeast two-hybrid screen was performed, identifying the protein WASH as a potential partner of Exo84 and Sec3. WASH is a Nucleation Promoting Factor (NPF) able to activate the actin nucleating Arp2/3 complex. Results of the present study showed that WASH and the exocyst complexes interact and localize on MT1-MMP-positive endosomes in MDA-MB-231 breast cancer cells. This study highlight a direct implication of WASH and exocyst complex in ECM degradation by cancer cells through the docking and exocytosis of MT1-MMP-containing endosomes at the PM through connections between these compartments and the extracellular medium. This WASH- and exocyst-dependent MT1-MMP exocytosis mechanism is required for degradation of adjacent tissue by cancer cells during tumour cell invasion.