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Spatial and temporal aspects of PI(4,5)P2 and SNAREs in exocytosis studied using isolated membrane sheets and capacitance measurements

dc.contributor.advisorNeher, Erwin Prof. Dr.de
dc.contributor.authorMilosevic, Irade
dc.date.accessioned2006-02-09T06:55:15Zde
dc.date.accessioned2013-01-18T14:28:39Zde
dc.date.available2013-01-30T23:51:11Zde
dc.date.issued2006-02-09de
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-0006-B5E6-7de
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-3296
dc.format.mimetypeapplication/pdfde
dc.language.isoengde
dc.rights.urihttp://webdoc.sub.gwdg.de/diss/copyr_diss.htmlde
dc.titleSpatial and temporal aspects of PI(4,5)P<sub>2</sub> and SNAREs in exocytosis studied using isolated membrane sheets and capacitance measurementsde
dc.typedoctoralThesisde
dc.title.translatedSpatial and temporal aspects of PI(4,5)P<sub>2</sub> and SNAREs in exocytosis studied using isolated membrane sheets and capacitance measurementsde
dc.contributor.refereeNeher, Erwin Prof. Dr.de
dc.date.examination2006-01-18de
dc.subject.dnb000 Allgemeinesde
dc.subject.dnbWissenschaftde
dc.description.abstractengCa2+-triggered exocytosis is a highly regulated process and the proteinaceous machinery mediating and regulating it has been studied in detail over the last decade. However, the participation of membrane lipids and their roles in this important process remain unclear. Therefore, in addition to studying the plasmalemmal members of a protein family central to the late steps of exocytosis, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors or SNAREs, I also explored the function of one plasmalemmal lipid, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P2.Exocytosis is organised spatially and temporally and my aim was to examine both these aspects of exocytic regulation with respect to SNAREs and PI(4,5)P2. To obtain the high temporal resolution, I used patch-clamp membrane capacitance and amperometric measurements while monitoring intracellular Ca2+-levels. To acquire the spatial information, I adapted the plasma membrane sheet technique: the spatial distribution and level of PI(4,5)P2 was studied using adult bovine cells and embryonic mouse chromaffin cells were employed to investigate that of SNARE proteins. The results I obtained using this powerful combination of electrophysiological and imaging assays are summarised in the following points:1) PI(4,5)P2, a signalling phospholipid with a dynamically fluctuating concentration, is spatially organised in the plasma membrane of chromaffin and pheochromocytoma cells. It is enriched in nanometric-sized clusters which require the presence of cholesterol. The PI(4,5)P2 clusters partially co-localise with SNARE protein syntaxin 1 clusters to which secretory vesicles preferentially dock and fuse.2) The level of PI(4,5)P2 in the inner leaflet of the plasma membrane can be semi-quantitatively measured and manipulated. Using a PI(4,5)P2-specific monovalent probe and the plasma membrane sheet assay, I detected a twofold increase in PI(4,5)P2 level upon overexpression of phosphatidylinositol-4-phosphate-5-kinase I- (PI4P5K-I-), and a nearly complete elimination of PI(4,5)P2 from the membrane upon overexpression of a membrane-targeted inositol 5´-phosphatase domain of synaptojanin 1 (IPP1-CAAX). I also determined a dual effect for the pharmacological drug LY294002 on PI(4,5)P2 abundance: short-term LY294002 application caused a twofold increase, while long-term LY294002 application resulted in a significant decrease of plasmalemmal PI(4,5)P2.3) PI(4,5)P2 is necessary for secretion, and moreover, its level directly controls the extent of exocytosis by regulating the releasable vesicle pool size but not the fusion kinetics. If PI(4,5)P2 is depleted from the plasma membrane by long-term LY294002 application or phosphatase overexpression, exocytosis is abolished or reduced, respectively. In contrast, when PI(4,5)P2 levels are increased by short-term LY294002 application, PI4P5K-I- overexpression or PI(4,5)P2 infusion, exocytosis is increased. Collectively, these findings imply that first, the cells do not compensate for a change in PI(4,5)P2 level, but employ it as an input signal to determine the extent of exocytosis; and second, in the resting chromaffin cells the PI(4,5)P2 level is limiting, so that an increase in PI(4,5)P2 concentration can up-regulate secretion. These features suggest that PI(4,5)P2 is a potential key physiological regulator of exocytosis.4) In a separate study, Gabor Nagy and I investigated the basis for the different secretory phenotypes associated with two SNAP-25 isoforms which differ by 9 amino acids. Using the plasma membrane sheet assay and chromaffin cells derived from SNAP-25 null mice, we found that both SNAP-25 splice variants are targeted to the plasma membrane with the same efficiency, are available in large excess and co-localise to the same extent with syntaxin 1 clusters. This allowed us then to investigate the role of the specific amino acid alterations in more detail. The results can be summarised as follows. The repositioning of one of the four palmitylated cysteines does not alter isoform targeting efficiency to exocytic sites. Systematic swapping of the three charge substitutions in the N-terminal SNARE domain was undertaken which indicated that two of the three non-conservative substitutions (H66Q, Q69K) are both necessary and sufficient to provide SNAP-25a with the secretory properties of SNAP-25b. The complementary substitution study in SNAP-25b revealed that the single K69Q mutation is sufficient to induce reversion to the SNAP-25a phenotype. If this is considered together with the observation that the Q69K mutation in SNAP-25 results in an intermediate phenotype, it implies that the amino acids in positions 66 and 69 do not have additive effects on exocytosis. Further biochemical and molecular dynamic stimulation experiments suggested that these two substitutions probably do not regulate exocytosis by affecting the SNARE complex properties, but rather they may play a role in the interaction with an accessory factor that could influence the stabilisation or formation of the primed vesicle state.5) Minor perturbations of SNAP-25 binding to its SNARE partners achieved by mutating residues within the interaction layers or through C-terminal deletion does not affect plasma membrane targeting or protein expression level. Jakob B. Sørensen was then able to use these mutated proteins to demonstrate that sequential N- to C-terminal zippering-up of the SNARE complex drives priming and fusion of secretory vesicles.de
dc.contributor.coRefereeJahn, Reinhard Prof. Dr.de
dc.contributor.thirdRefereeSchild, Detlev Prof. Dr.de
dc.subject.topicMolecular Biology & Neurosciences Programde
dc.subject.engexocytosisde
dc.subject.engPI(4de
dc.subject.eng5)P<sub>2</sub>de
dc.subject.engSNAREde
dc.subject.engchromaffin cellde
dc.subject.engSNAP-25de
dc.subject.engvesiclede
dc.subject.engelectrophysiologyde
dc.subject.engplasma membranede
dc.subject.engmicrodomainsde
dc.subject.engphospholipidde
dc.subject.engcapacitancede
dc.subject.engamperometryde
dc.subject.engmicroscopyde
dc.subject.engknock-outde
dc.subject.engsyntaxinde
dc.subject.engexocytosisde
dc.subject.engPI(4de
dc.subject.eng5)P<sub>2</sub>de
dc.subject.engSNAREde
dc.subject.engchromaffin cellde
dc.subject.engSNAP-25de
dc.subject.engvesiclede
dc.subject.engelectrophysiologyde
dc.subject.engplasma membranede
dc.subject.engmicrodomainsde
dc.subject.engphospholipidde
dc.subject.engcapacitancede
dc.subject.engamperometryde
dc.subject.engmicroscopyde
dc.subject.engknock-outde
dc.subject.engsyntaxinde
dc.subject.bk42.00 Biologie: Allgemeinesde
dc.subject.bk42.17 Allgemeine Physiologiede
dc.subject.bk42.12 Biophysikde
dc.subject.bk42.15 Zellbiologiede
dc.identifier.urnurn:nbn:de:gbv:7-webdoc-656-8de
dc.identifier.purlwebdoc-656de
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullWCK 000 Elektrophysiologiede
dc.subject.gokfullWCC 000 Molekulare Biophysik. Biophysikalische Chemiede
dc.subject.gokfullWF 100 Methodende
dc.identifier.ppn515217557de


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