Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Studies that replace or mutate the TM domain of the F protein of several viruses implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was used to demonstrate that isolated TM domains of HeV F protein associate in a monomer-trimer equilibrium. To determine factors driving this association, we analyzed the sequence of several paramyxovirus F protein TM domains and found a heptad repeat of β-branched residues. Analysis of the HeV F TM domain specifically revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein expression and pre-fusion conformation. To further understand the role of the TM domain, the TM domain was targeted as a potential modulator of F protein stability and function. Exogenous HeV F TM constructs were co-expressed with the full length F protein in Vero cells to analyze the effects on protein expression. Co-expression of the exogenous HeV F TM constructs dramatically reduced the expression of HeV F. However, the co-expression of exogenous HeV F TM constructs with a different paramyxovirus F protein, PIV5 F, did not strongly affect PIV5 F expression levels, suggesting that the interaction of the exogenous TM constructs is specific. Fusion assays revealed that HeV F TM constructs dramatically reduced HeV F, but not PIV5 F fusion activity. We hypothesize that the short exogenous HeV TM constructs associate with the TM domain from full-length HeV F, resulting in pre-mature triggering or protein misfolding. The work presented here demonstrates that specific elements in the TM domain contribute to TM association and pre-fusion protein stability. Furthermore, targeting these interactions may be a viable approach for antiviral development against this important pathogen.

No description available.

The Chediak-Higashi Syndrome is a disorder affecting lysosome biogenesis. At the cellular level, the Chediak-Higashi syndrome is characterized by the presence of grossly enlarged lysosomes in every tissue. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. The Chediak-Higashi syndrome is caused by the loss of a BEACH protein of unknown function named Lyst. In this work, I have studied the function of the Dictyostelium LvsB protein, the ortholog of mammalian Lyst and a protein that is also important for lysosomal function. Using a knock-in approach we tagged LvsB with GFP and expressed it from its single chromosomal locus. GFP-LvsB was observed on endocytic and phagocytic compartments. Specific analysis of the endocytic compartments labeled by LvsB showed that they represented late lysosomes and postlysosomes. The analysis of LvsB-null cells revealed that loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. This work demonstrated that the abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of lysosomes with postlysosomal compartments. Furthermore, this work provided the first evidence that LvsB is a functional antagonist of the GTPase Rab14 in vesicle fusion events. In particular, we demonstrated that reduction of Rab14 activity suppressed the LvsB-null phenotype by reducing the enlarged post-lysosomes and the enhanced rate of heterotypic fusion. In contrast, expression of an active form of Rab14 enhanced the LvsB-null phenotype by causing an even more severe enlargement of endosome size. The results provided by this work support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early with late compartments and antagonize Rab GTPases in membrane fusion. The LvsB localization studies and the functional assessment of the LvsB-null phenotype helped make unique contributions to the understanding of the molecular function of Lyst proteins.

FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogeneis. To investigate the role of FER-1 in membranous organelle fusion, I first examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains which commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. I found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which may arise from FER-1 by proteolysis. By both light and electron microscopy these FER-1 proteins are localized to membranous organelle membranes. Together, these results suggest that the ferlin family members may share a conserved mechanism to regulate cell-type specific membrane fusion.In Chapter III, I present additional results toward studying the function of FER-1 using several broad-based approaches. First, I present a bioinformatics analysis of FER-1 C2 domains and the preliminary results of their calcium-dependent phospholipid binding capabilities. Second, preliminary interactions found with individual FER-1 functional domains by a yeast-two hybrid screen are discussed. Lastly, I present results from a candidate-gene approach to identify additional regulators of MO fusion, the sperm-specific synaptobrevins.

No description available.

Given the debilitating effect that mitochondrial dysfunction has on human health, it is important to understand mitochondrial dynamics that are vital for the maintenance of mitochondrial function, genome, morphology, and quality control. Mitochondrial dynamics result from a balance in mitochondrial fusion and fission. Although the mechanism and regulation of mitochondrial fission are largely elucidated, less is known about mitochondrial fusion. Mgm1 is a protein that mediates mitochondrial fusion in yeast. However, the molecular mechanism of Mgm1 function in mediating mitochondrial fusion is unclear. In this thesis, first, I show that Mgm1 contains a lipid-binding domain by demonstrating that purified Mgm1 has lipid-binding activity and by identifying mutations in conserved residues that abrogate these interactions. Second, I show that Mgm1 assembles into hexameric rings and undergoes nucleotide-dependent structural transitions that, I believe, initiate membrane fusion. Lastly, I demonstrate that Mgm1 exhibits membrane-remodeling activities that are crucial for the tethering and lipid-mixing steps in the membrane fusion event. Together, I propose a mechanistic model of Mgm1 function in mediating mitochondrial fusion that advances the fields of mitochondrial biology, cellular protein-membrane dynamics, and the etiology of neurodegenerative diseases.

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 (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (PhD) - Swinburne University of Technology, 2008. / Submitted for the degree of Doctor of Philosophy, Swinburne University of Technology - 2008. Typescript. Includes bibliographical references (p. 195-208).

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references.