Hannover, Universiẗat, Diss., 2003.

Heidelberg, University, Diss., 2003.

Hannover, Universiẗat, Diss., 2003.

No description available.

Neurotransmission is essential for neuronal communication. At the presynapse, synaptic vesicles (SVs) undergo exocytosis to release neurotransmitter in response to incoming action potentials, and endocytosis to maintain the supply of SVs needed for further rounds of exocytosis. A key event during SV endocytosis is the efficient sorting and localisation of SV proteins at the plasma membrane. This ensures that nascent SVs that are formed have the correct molecular composition to participate in subsequent exocytic events. The sorting of SV proteins at the plasma membrane is usually facilitated by adaptor proteins (e.g. AP-2) which recognise binding motifs present on key SV proteins and facilitate their internalisation during endocytosis. In addition to this, certain SV proteins possess the ability to chaperone each other as part of an endocytic transport complex throughout the SV recycling process. In conjunction with AP-2-facilitated sorting, the transport of complexed SV proteins during endocytosis provides further mechanistic insight into how SVs are generated with consistent high fidelity for functional viability. Using pHluorins as a tool to visualise SV protein trafficking in hippocampal cultures, the relationship between two key SV proteins, synaptic vesicle protein 2A (SV2A) and synaptotagmin I (SYT1), was investigated. SYT1 predominantly acts as the Ca2+ sensor for fast synchronous release at the presynapse, whilst the exact function of SV2A remains unknown to this day. In this study, the ablation of the AP-2 binding site in SV2A (Y46A) resulted in increased SYT1 surface expression and accelerated SYT1 retrieval compared to WT SV2A. No additive defects were observed when a second point mutation (T84A) was introduced to SV2A that disrupts the phosphorylation-dependent interaction between SV2A and SYT1, thus confirming that SYT1 localisation and retrieval is dependent on normal SV2A retrieval by AP-2. The hypothesis that disruption of the SV2A-SYT1 interaction may provide an underlying mechanism for motor onset seizures in epilepsy was also investigated. An epilepsy-related mutation (R383Q) in SV2A also resulted in increased SYT1 surface expression and accelerated SYT1 retrieval mirroring the defects caused by the Y46A mutation. Introduction of Y46A or T84A mutation into SV2A R383Q resulted in no additive defects compared to the single mutant, suggesting that the observed defects in SYT1 localisation and retrieval kinetics in the epilepsy-related mutant may be caused by the ablation of normal SV2A internalisation. GST pulldown assays, mass spectrometry and western blotting data indicate that presence of the mutation disrupts normal binding of the SV2A cytosolic loop with actin, tubulin and certain subunits of V-ATPase. Finally, a link between SV2A-dependent presynaptic dysfunction and epilepsy was examined through studies utilising the anti-epileptic drug, levetiracetam (LEV). SV2A contains a binding site for LEV, suggesting that it may act as a carrier for the drug into the presynapse. Hippocampal neuronal cultures were treated with LEV at various concentrations in the presence of specific patterns of neuronal activity. No observed effects of the drug on synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and SYT1 recycling were observed, suggesting that LEV is unlikely to function as a modulator of excitatory presynaptic activity or by influencing SV2A function. In conclusion, this work demonstrates that SV2A is essential for accurate SYT1 trafficking and a link has been established between defective SV2A internalisation and subsequent downstream effects on SYT1 localisation and retrieval during SV recycling.

Sculpting membranes into dynamic, curved shapes is central to intracellular cargo trafficking and other cellular functions. However, generation of membrane curvature during trafficking involves lipids and membrane-associated proteins; current mechanisms focus on creating rigid cages, curved scaffolds, or membrane surface area changes by proteins. This dissertation provides an alternative mechanistic example for the control of membrane deformations, involving modulation of membrane material properties. Sar1, a GTPase of the COPII family, regulates vesicle trafficking from the endoplasmic reticulum. We find that Sar1p lowers the rigidity of the lipid bilayer membrane to which it binds. We examine the behavior of Saccharomyces cerevisiae Sar1 (Sar1p) and Homo sapiens paralogs of Sar1 (Sar1A and Sar1B). Like Sar1p, human Sar1s lower membrane rigidity. Unlike Sar1p, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which human Sar1 clustering governs membrane mechanical properties. Membrane rigidity measurements remain rare, however, and show a large variance, a situation that can be addressed by improving techniques and comparing disparate techniques applied to the same systems. I introduce applying selective plane illumination microscopy (SPIM) to image thermal fluctuations of giant vesicles. SPIM's optical sectioning enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, we show rigidity values from giant unilamellar vesicle fluctuations in close agreement with those derived from our independent assay based on membrane tether pulling. We also show that a model of homogeneous quasi-spherical vesicles poorly fits fluctuation spectra of vesicles bound by Sar1A at high concentrations, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties. I conclude by discussing our current work on amphipathic alpha helices, their ability to reduce membrane rigidity, and our hopes to create artificial helical structures capable of mimicking trafficking systems. Supplemental videos represent membrane disintegration with Sar1p and fluctuations of membrane only and Sar1p incubated vesicles. This dissertation contains previously published co-authored material.

No description available.

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.

No description available.

Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.