Phospholipid liposome binding to hydrogel beads based on poly(N-isopropylacrylamide) (PNIPAM) is accomplished employing either avidin/biotin conjugation or hydrophobic modification of the microgels, and the ability to form single supported lipid bilayers is explored. The co-monomer acrylic acid (AA), evenly distributed or localized to the shell of the microgel, is included to facilitate post-polymerization chemical modification of the hydrogel beads. The degree of chemical modification of the microgels as well as the thermal behavior is monitored via 1H and 13C nuclear magnetic resonance (NMR). Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phoshocholine (POPC) and a small amount of commercially available biotinylated-lipid are shown to bind as intact entities while sequestering internal contents to biotinylated hydrogel beads, utilizing avidin as the coupling agent. Under fusogenic conditions, these bound liposomes remain as individual vesicles. Alternatively, POPC liposomes are shown to bind to microgels modified to display single chain alkyl groups or cholesteryl moieties, and remain as intact vesicles. It is demonstrated that these liposomes become permeable at high hydrophobe content. Bound liposomes will fuse into larger structures under high hydrophobe content conditions, but remain permeable. The volume phase transition (VPT) characteristic of PNIPAM microgels is shown to influence the permeability of hydrophobically bound (low hydrophobe content), but not avidin/biotin conjugated, liposomes. The degree of liposome binding, as well as their resulting structures and permeability are investigated utilizing 31P NMR, fluorescence spectroscopy and microscopy. The microgel-bound liposome and microgel-supported lipid bilayer hybrid systems would be ideally suited to drug delivery and tissue engineering applications. The microgel-supported single lipid bilayer system would, in addition, potentially act as a cell model system for membrane dynamics and embedded amphiphile NMR studies.

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [ H]NE uptake capacity, an effect characterized by a robust decrease in the V of the transport of [ H]NE. As expected, reserpine did not displace the binding of [ H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca /Ca -calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [ H]NE uptake and eliminated the inhibitory effects of reserpine on [ H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca -independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

No description available.

Synaptophysin (Syp) is a major integral synaptic vesicle (SV) protein; there are 31 copies of Syp per vesicle, which totals up to 10% of the total SV protein content. Despite being the major SV protein, little is known about the interaction partners of Syp and as a result there has been no clear role attributed to it. One key feature of Syp is that its cytoplasmic C-terminus contains 10 pentapeptide repeats, nine of which are initiated by a tyrosine residue. Syp is the major tyrosine phospho-protein on SVs. The kinase thought to phosphorylate Syp in vivo is the ubiquitously expressed non-receptor kinase C-Src. There are two splice variants of C-Src, N1- and N2-Src, which are only expressed in neuronal tissues. Although the 3 Srcs are structurally similar, they differ by a small insert of amino acids into their SH3 domains (the N-Src loop). Examination of the amino acid sequence of the cytosolic C-terminus of Syp revealed a putative type one SH3 domain interaction motif. A screen using SH3 domains of synaptic proteins as bait in GST-pull downs from nerve terminal lysate allowed an inventory of potential interaction partners of Syp to be created. Reciprocal experiments using the C-terminal of Syp as bait confirmed many of these interactions. Single point mutations of the SH3 interaction motif on Syp highlighted that syndapin and C-Src bound to Syp via this motif. These binding mutants were inserted in Syp superecliptic synaptophluorin (SypHy) to determine the functional consequences of these interactions. These mutants did not affect the trafficking of Syp when expressed in cortical neurons derived from Syp knockout mice. However, the SH3 interaction motif was fundamental for the retrieval of VAMP (vesicle associated membrane protein) when expressed in Syp knockout cultures. Importantly, this role is not mediated through a direct interaction with VAMP with the SH3 interaction motif implicating either syndapin, C-Src or both in Syp-dependent VAMP retrieval. The 3 different Srcs had different methods of interaction with Syp, and in vitro protein kinase assays the ability of the three Src splice variants to phosphorylate Syp was assessed. Key differences in both speed and efficiency of Syp phosphorylation was observed for the different Src splice variants. Mutagenesis of either all 9 tyrosine residues, only previously identified sites resulted in changes in Syp interactions in GST-pull down assays from nerve terminal lysates. To investigate the role of Syp phosphorylation in the SV lifecycle, the tyrosine pentapeptide repeats were truncated from the C-terminal of Syp in both a mCerulean tagged Syp and SypHy. The experiments showed that these potential tyrosine phosphorylation sites were not involved in the trafficking of Syp but key in the retrieval of VAMP from the plasma membrane during the SV lifecycle. I have indentified an SH3 interaction motif on the C-terminal of Syp that is critical in forming a complex of proteins that are responsible for the retrieval of VAMP during the SV lifecycle. Further experiments have shown that this key interaction is potentially phosphorylation dependent. My preliminary mass spectrometry analysis has provided a catalogue of proteins that can potentially interact with Syp, identifying proteins that may bind to either the Syp C-terminus SH3 interaction motif or to other regions in a phosphorylation dependent manner. This has provided a list of potential candidate proteins for the VAMP retrieval complex.

The crayfish claw opener neuromuscular junction (NMJ) is a biological model for studying presynaptic neuromodulation by serotonin and synaptic vesicle recycling. Serotonin acts on crayfish axon terminals to increase the release of the neurotransmitter glutamate, but a complete understanding of its mechanisms of action are unknown. In order to sustain enhanced neurotransmission over long periods of time, it was hypothesized that serotonin recruits (activates) a population of previously non-recycling vesicles to become releasable and contribute to neurotransmission. To determine if serotonin activates a distinct population of synaptic vesicles, FM1-43 fluorescence unloading experiments were performed on crayfish excitatory opener axon terminals. These experiments could not resolve a serotonin-activated population of synaptic vesicles, but instead revealed that synaptic vesicles change behaviour in axon terminals independent of serotonin, with vesicles becoming less likely to exocytose and unload FM1-43 dye over time. The change in behaviour was hypothesized to be due to conversion of vesicles from a recycling (releasable) status to a reserve (reluctant to release) status. Synaptic vesicle pool localization was then tested using photoconversion of FM1-43 and transmission electron microscopy techniques. The spatial location of FM1-43-labeled vesicles fixed 2 minutes following 20 Hz stimulation did not reveal retention of vesicles specifically near release sites and the distribution of FM1-43-labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 seconds following stimulation, however, contained numerous endosome-like structures - the most frequently observed structures resembled large vesicles, which were the equivalent of 2-5 regular vesicle sizes. These results suggest that crayfish axon terminals recycle vast amounts of membrane in response to sustained 20-Hz stimulation and endocytosis appears to occur via multiple routes with the most common being through large vesicle intermediates. / Graduate

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This project focuses on the stabilisation, modification, delivery and treatment of phospholipid based vesicles for applications in advanced wound care, with a focus on paediatric burns. Vesicles, commonly referred to as liposomes or nanocapsules, are attractive drug delivery composites, due to their biocompatible properties. They have the ability to entrap active compounds within their core, which can be released at the point of use, (in vivo or ex vivo) either through passive diffusion, or in response to local environmental stimulus.

<p>A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.</p>