The immune system is comprised of specific cell types that communicate and interact via a range of soluble and surface-bound molecules to defend the body against pathogens. Many gaps remain in our understanding of the subcellular trafficking pathways that regulate the diverse functions of the immune system. The central aim of this thesis was to investigate transport through the endocytic pathway, focussing in particular on the unique organelles and functions of this pathway in immune cells. Two subsets of immune cells were of particular interest in this thesis, macrophages and natural killer (NK) cells. These cell types both perform a range of functions that contribute to both innate and adaptive immunity. Another common thread between these cells is that they both perform functions involving specialised endocytic organelles and pathways. Macrophages utilise their endocytic pathways to perform several unique functions; phagocytosis, endocytosis and degradation of foreign proteins for presentation on MHC class II molecules, and signalling of Toll-like receptors from endosomes. Even secretion of cytokines such as tumour necrosis factor alpha (TNFα) by macrophages requires transport through an endosomal compartment, the recycling endosome, as recently discovered in this laboratory (Murray et al., 2005a). NK cells utilise specialised secretory lysosomes to deliver a lethal hit to carefully identified target cells, providing an alternative example of specialised endocytic trafficking in the immune system. Of the many protein families that regulate subcellular trafficking, the SNARE, Rab, Munc and exocyst proteins were focussed on during this thesis. The localisation and function of members of these families in the endocytic pathway were investigated. Novel results in macrophages concerned the role of Vti1b in endocytosis, a process with implications for MHC class II antigen presentation and TLR detection of endocytosed particles. Alteration of Vti1b protein levels in the cells significantly decreased uptake and degradation of endocytic cargo. A role for Rab11 and the recycling endosome in antigen presentation was also studied. MHC class II was detected in recycling endosomes, and overexpression of a mutant Rab11 protein altered the distribution of MHC class II, suggesting a role for Rab11 in subcellular trafficking during antigen presentation. Preliminary results also suggest a role for the exocyst protein Sec15 at the recycling endosome in macrophages, providing a new target for investigation of the regulation of TNFα secretion. The recycling endosome is emerging as a vital transport hub during cytokine secretion, phagocytosis and possibly other cellular functions in macrophages. This project also involved the unique opportunity to examine primary NK cells from patients with a number of genetic immunodeficiencies caused by mutations to trafficking proteins. The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial haemophagocytic lymphohistiocytosis types 3 (FHL3) and 4 (FHL4) are associated with loss-of-function of Rab27a, Munc13-4 and syntaxin 11 (Stx11), respectively. These diseases involve a loss of cytotoxic function by cytotoxic CD8+ T lymphocytes and NK cells, but the precise molecular role of these proteins in granule release is incompletely understood. In freshly isolated, resting NK cells from healthy subjects, PMA and ionomycin stimulation or conjugation to susceptible target cells induced colocalisation of endogenous Rab27a and Munc13-4 to perforin-containing granules. In Rab27a-deficient cells, which showed defective degranulation and cytotoxicity induced by signals for both natural and antibody-dependent cellular cytotoxicity, Munc13-4 failed to colocalise with perforin upon activation. Unexpectedly, Rab27a and Munc13-4 localisation to lytic granules was selectively induced by different receptor signals, demonstrating specificity for regulation of lytic granule maturation by target cell ligand expression. Recruitment of the SNARE protein Vti1b, which has not previously been associated with NK cell function or secretory lysosome release, to perforin granules was also discovered. Unexpectedly, Stx11 was not localised to perforin granules. These experiments have contributed to our understanding of the precise molecular roles of Munc13-4, Rab27a and Stx11 in NK cell granule release. Overall, this thesis presents novel and important results from studies of subcellular transport through the endocytic pathways of macrophages and NK cells. These results advance our understanding of several immune functions, and a number of human genetic immunodeficiencies. This new knowledge of the role of endocytic organelles and fusion machinery in these processes provides exciting targets for future research.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 80-88

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.

This dissertation discusses the potential of the snare drum as a solo concert instrument. Four pieces from a collection entitled The Noble Snare are used for demonstration ("Homily" by Milton Babbitt, "Composed Improvisation for Snare Drum" by John Cage, "Peeping Tom" by Dan Senn, and "The Noble Snare" by Stuart Saunders Smith). In the absence of many traditional musical devices (i.e. melody and harmony), alternative means of expression are used by the composer. Each piece is discussed with regard to its distinctive compositional approach and inherent performance issues. Information is also given pertaining to the background of the Noble Snare series. This includes: the inspiration for the project, editorial issues, and its influence on snare drum performance. Much of this research was completed through interviews by with author with Sylvia Smith, publisher of The Noble Snare and owner of Smith Publications.

Plants have an artillery to defend themselves. The plant surface is protected by water- resistant cuticle and mechanically strong cell wall. Then each plant cell has tools to recognize and to answer to a pathogen threat. In an extreme case, the answer is programmed cell death. Plant immunity is a complex process integrating these passive and active mechanisms in an effort to overstay a pathogen attack. When the plant cell is attacked by a pathogen, the metabolic resources are redirected towards immunity reaction which results in growth restriction. Both the immunity reaction and the growth are dependent on the efficient polarized secretion of various cargoes. Exocyst complex mediates tethering of a secretory vesicle with a target membrane and SNARE complex orchestrates the subsequent steps of vesicle docking and fusion. Exocyst and SNAREs are regulated by various proteins. In my work, I focused on identifying the exocyst interaction partners in plant immunity. In cooperation with my colleagues, we found the direct association between Qa-SNARE SYP121 involved in plant penetration resistance and EXO70B2 exocyst subunit. Moreover, we confirmed the relevance of their interaction for the formation of epidermal defensive structures, papillae and haustorial encasements in plant defence against non-adapted...

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes involves defects in glucose-stimulated insulin secretion (GSIS) from the pancreatic beta cells in combination with defects in peripheral (muscle and adipose) tissue glucose uptake. Both GSIS and glucose uptake are regulated by Syntaxin 4 (Syn4)-Munc18c complexes. Importantly, reports link obesity and Type 2 diabetes in humans with changes in protein levels of Munc18c and Syn4; yet the molecular mechanisms underlying this requirement remain unclear. The central hypothesis proposed is that Syn4-Munc18c complexes are modulated by post-translational modifications and novel interactions. Toward this, we found that Syn4-Munc18c complexes are regulated by tyrosine phosphorylation of Munc18c at Y219 in beta cells. Munc18c tyrosine phosphorylation disrupts Syn4-Munc18c complexes, which leads to an increase in Munc18c associating with the double C2 domain protein Doc2β. Disruption of Syn4-Munc18c upon tyrosine phosphorylation results in an increase in Syn4-SNARE complex formation and GSIS from beta cells. Similarly, tyrosine phosphorylation of Munc18c at Y219 and also Y521, disrupts its association with Syn4 in insulin-stimulated 3T3L1 adipocytes and skeletal muscle. In vitro kinase assays further suggested that the insulin receptor tyrosine kinase targeted Y521 of Munc18c. Further investigations using 3T3L1 adipocytes and skeletal muscle extracts indicate that Munc18c interacts with the insulin receptor tyrosine kinase in an insulin-dependent manner, resulting in phosphorylation of Munc18c, coordinate with the timing of its dissociation from Syn4. Finally, we found that stimulus-induced changes occurred also with Syn4, most notably in the islet beta cells. Syn4-mediated insulin release requires F-actin remodeling to mobilize insulin granules to the plasma membrane. Our studies reveal that Syn4 directly associates with F-actin in MIN6 beta cells, and that the disruption of this complex correlates with increases in glucose-stimulated insulin secretion. Future studies will focus upon the potential link between Syn4, F-actin remodeling with Munc18c, to further gain understanding of the requirements for Syn4-Munc18c complexes in insulin secretion. In sum, given the parallels of Munc18c tyrosine phosphorylation in regulating Syn4-Munc18c interaction and exocytosis in beta cells and peripheral tissues, manipulations of this complex may have therapeutic potential as a strategy to treat Type 2 diabetes.

Plants have an artillery to defend themselves. The plant surface is protected by water- resistant cuticle and mechanically strong cell wall. Then each plant cell has tools to recognize and to answer to a pathogen threat. In an extreme case, the answer is programmed cell death. Plant immunity is a complex process integrating these passive and active mechanisms in an effort to overstay a pathogen attack. When the plant cell is attacked by a pathogen, the metabolic resources are redirected towards immunity reaction which results in growth restriction. Both the immunity reaction and the growth are dependent on the efficient polarized secretion of various cargoes. Exocyst complex mediates tethering of a secretory vesicle with a target membrane and SNARE complex orchestrates the subsequent steps of vesicle docking and fusion. Exocyst and SNAREs are regulated by various proteins. In my work, I focused on identifying the exocyst interaction partners in plant immunity. In cooperation with my colleagues, we found the direct association between Qa-SNARE SYP121 involved in plant penetration resistance and EXO70B2 exocyst subunit. Moreover, we confirmed the relevance of their interaction for the formation of epidermal defensive structures, papillae and haustorial encasements in plant defence against non-adapted...

Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.

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