Regulation of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNARE) mediated exocytosis is dependent upon four key proteins; the vesicular SNARE synaptobrevin, target SNAREs SNAP-25 and syntaxin and the Sec1/Munc18 (SM) protein munc18-1. Despite the munc18-1-syntaxin interaction being central to regulated vesicle exocytosis the spatial and temporal pattern of their molecular distribution and interaction in neuroendocrine and neuronal cells remains undefined. Using in vitro and molecular approaches this thesis shows that disruption of the munc18- 1-syntaxin-N-terminal interaction results in significant changes in syntaxin localisation, membrane-proximal vesicle dynamics and fusion efficiency within neuroendocrine cells. Using the super-resolution techniques Ground State Depletion-Individual molecule return (GSDIM) Microscopy and Photoactivation Localisation Microscopy (PALM) this thesis has demonstrated that the spatial distribution of single munc18-1 molecules is non-random and that few munc18-1 molecules are required for exocytosis to proceed in neuroendocrine cells. Furthermore, targeted disruption of the N-terminal interaction resulted only in a reorganisation of interaction with syntaxin with no change in the molecular spatial pattern of secretory vesicles, syntaxin or munc18-1. Single molecule imaging PALM (sptPALM) enabled the investigation of the complex spatio-temporal behaviours of single munc18-1 molecules in living neuroendocrine cells. Spatially resolved maps of single munc18-1 molecules demonstrated that munc18-1 exhibits a caged motion within areas of the plasma membrane and were found to move between molecular storage depots distinct from vesicle docking sites. To explore the precise spatial and temporal sequence of interactions between syntaxin and munc18-1 in living neurons, super-resolution imaging techniques PALM and sptPALM were employed. Two kinetically and spatially distinct populations of munc18-1 molecules co-exist within a living neuron and munc18-1 requires syntaxin to traffic efficiently in axons but not for its retention in nerve terminals. Moreover, Fluorescence Correlation Spectroscopy (FCS) revealed that the majority of munc18-1 molecules do not interact with syntaxin in nerve terminals and the diffusion rate of syntaxin is significantly slowed down upon neuronal depolarisation.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1999. / Includes bibliographical references. Also available in the Internet.

Thesis (Ph.D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 24, 2009) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2005. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 76 - 80). Also available for download via the World Wide Web; free to University of Oregon users.

No description available.

Many studies in different eukaryotes have shown the importance of the vesicle-tethering exocyst complex for cellular processes dependent on intensive polarized secretion. The plant exocyst complex is crucial for regulation of cell polarity, morphogenesis, and defence. In land plants, gene encoding the EXO70 exocyst subunit multiplied into many paralogs, but only a few of them have been functionally described. In this thesis, the EXO70A2 isoform, a member of the EXO70.1 subfamily, was found to be the main EXO70 exocyst subunit involved in the canonical function of the exocyst complex in Arabidopsis pollen. EXO70A2 is important for several stages of pollen development-pollen grain maturation, germination, and pollen tube growth. Pollen-expressed EXO70A2 was the only EXO70 isoform able to substitute for the function of EXO70A1 in the sporophyte, but not vice-versa. This indicates partial functional redundancy of these two closely related isoforms and a high specificity for pollen-related processes. The finding that the exocyst is targeted to the plasma membrane via EXO70A1 subunit is further elaborated in the thesis. EXO70A1 binds plasma membrane via interactions with specific phospholipids that form a unique plasma membrane-lipid signature in plants. Other isoform, EXO70B1 from the EXO70.2 subfamily,...

Exocytosis, the fusion of intracellular vesicles with the plasma membrane, is fundamental to intercellular communication in multicellular organisms. This pathway facilitates the release or secretion of molecules from the cell. In addition, exocytosis is essential for delivery of resident proteins to the plasma membrane. There are two different pathways of exocytosis, constitutive and regulated exocytosis. Constitutive exocytosis occurs without regulation, e.g. pathways regulating the delivery of lipids and ‘house-keeping’ proteins to the plasma membrane or the secretion of antibodies and extra-cellular matrix components from the cell. In contrast, regulated exocytosis facilitates the controlled release of extra-cellular molecules or insertion of new membrane components only in response to a physiological signal. The most common signal for regulated exocytosis is an increase in intracellular Ca2+ concentration. Several proteins function in exocytosis, and the membrane fusion step is widely believed to result from an interaction between SNARE (SNAP receptor) proteins on the vesicle membrane and plasma membrane. In neuroendocrine cells, these SNARE proteins are VAMP2, which is bound to vesicle membranes and syntaxin1A and SNAP25, which are associated with the plasma membrane. Several proteins have been implicated as SNARE regulators, such as NSF (N-ethylmaleimide-sensitive factor) and its cofactor α-SNAP, Munc18 and synaptotagmin. Another possible SNARE regulator is the cysteine string protein (CSP). CSPα was first identified in Drosophila melanogaster and was later identified in Torpedo as a possible Ca2+-channel regulator. Inactivation of the CSPα gene in Drosophila is lethal at an embryonic stage and in embryos synaptic vesicle exocytosis was decreased by ~50% at 22°C and was abolished at higher temperatures. These results provided strong evidence that CSPα has an important role in presynaptic neurotransmission. However, more recent work on CSPα null mice uncovered an important neuroprotective function for CSPα in brain, but also challenged the proposed function of CSPα in neuronal exocytosis, as no defect in this pathway was evident, at least in young animals. The only reported developmental abnormality of CSPα null mice was bilateral cryptorchidism, a failure of testicular descent during development. Interestingly, two additional CSP isoforms were recently identified in mouse and human testis, CSPβ and CSPγ. One consequence of the identification of CSPβ and CSPγ is that they may complicate analysis of CSPα knockout mice. Here, we have used a combination of techniques, cell systems and human brain samples to examine the function of CSPα in exocytosis, the expression of novel CSPα isoforms in testis, and expression changes of CSPα and its partner proteins in neurological disorders. Furthermore, we have initiated studies to examine how CSPα function is linked to cryptorchidism at the molecular level. My results show that CSPα depletion perturbs regulated exocytosis in neuroendocrine cells, but has no consistent effect on constitutive exocytosis. CSPα has been reported to have an important neuroprotective function; however, no significant changes in CSPα expression were detected in brain samples for schizophrenia, depression and bipolar disorder. Nevertheless the expression of specific CSPα binding partners was found to be significantly changed in some of these disorders. In addition to these studies focussing on CSPα function and expression in neuronal and neuroendocrine cells, studies were undertaken to analyse expression profiles of CSP isoforms in testis. This analysis found that CSPβ and CSPγ are exclusively expressed in testis, and that mRNA transcription of both isoforms is initiated with sexual maturation. Furthermore expression of both isoforms is restricted to germ cells, whereas CSPα is expressed throughout testes. Previous work has shown that the secretory hormone INSL3, which is exclusively expressed in testicular Leydig cells, is involved in the development of cryptorchidism. Confocal microscopic analysis revealed that CSPα and INSL3 colocalise on vesicles in Leydig cells, suggesting the intriguing possibility that CSPα inactivation might cause cryptorchidism due to a loss of INSL3 secretion.

No description available.

Src is a 60 kDa tyrosine kinase that is expressed in most of animal tissues. Src has three splice variants, C-src, which is ubiquitously expressed, and N1- and N2-src, which are neuronal specific splice variants. The srcs are differentially spliced at their SH3 domains, therefore the hypothesis is that this splicing allows them to have different binding partners and perform different roles in neurons. The aim of this project is to identify new interactions for the three src splice variants in neurons and their possible functional roles. The SH3 domains, kinase active truncated proteins ( 80) and kinase dead mutant full length versions of the three splice variants of src were cloned from a rat brain cDNA library into bacterial expression vectors. GST-pull downs from nerve terminal lysates showed that different src splice variants had different binding partners. These partners were identified by mass spectrometry and confirmed by western blotting. C-src binding partners included dynamin, synapsin, and synaptojanin, while N2-src binding partners included synaptophysin, Munc18-1, and NSF. The interaction between N2-src and Munc 18-1 was characterized further; however a number of in vitro interaction assays and kinase assays showed that Munc 18-1 may not be a direct binding partner for N2-src or substrate. N1-src displayed a stimulation-dependent interaction with dynamin I. This was shown to be phosphorylation-dependent in contrast to C-src binding. The major phosphorylation sites on dynamin I, S774 and S778, were not involved in the regulation of N1-src binding. The binding site for N1-src on dynamin I was different to C-src, with extensive mutagenesis studies suggesting that the interaction site is at the tail of the dynamin I xa splice variant, which has an additional two phosphorylation sites.

Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.