Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T03:25:29Z (GMT). No. of bitstreams: 1 Cunha_DanielAndradeda_D.pdf: 2004407 bytes, checksum: 40e252218ab6f8007c9bfb5afde02a52 (MD5) Previous issue date: 2006 / Resumo: Prolactina induz a maturação da resposta secretória das células B pancreáticas em ilhotas de ratos neonatos in vitro. Assim, o objetivo deste trabalho foi avaliar se a maturação na resposta a glicose, induzida pela prolactina, está associada a alterações na expressão, associação e fosforilação de proteínas envolvidas na mobilização e extrusão dos grânulos de insulina. Para isso, ilhotas pancreáticas de ratos neonatos foram cultivadas por cinco dias em presença de prolactina e após extração do RNA e proteína total foram realizados RT-PCR e western blot. Observamos aumento na expressão gênica e protéica da MAP-2 e cinesina em ilhotas cultivadas com prolactina. Analisamos também a associação e fosforilação através de imunoprecipitação seguido de western blot de proteínas SNARE e MAP-2/cinesina em ilhotas estimuladas agudamente (20 min) com prolactina. Prolactina aumentou a associação entre proteínas SNARE e MAP-2/cinesina e reduziu a ligação entre sintaxina IA/munc-I8. Fosforilação em resíduos serina das proteínas SNAP-25, sintaxina IA, munc-I8 e MAP-2 encontravam-se aumentadas enquanto que da cinesina foi diminuída, em ilhotas estimuladas com prolactina. Ainda, foi observado aumento na formação do complexo SNARE em ilhotas agudamente estimuladas com prolactina, 22 mM de glicose, 40 de mM K+, 200 J.lMde carbacol e I J.lMde PMA (ativador da PKC). A inibição da via da MAP cinase, por PD098059, bloqueou a formação do complexo SNARE e fosforilação da sintaxina induzida por prolactina. Desta forma, podemos concluir que prolactina auxilia na maturação das células B por aumentar a expressão, fosforilação e associação de proteínas que compõem a maquinaria de extrusão dos grânulos de insulina, provavelmente via MAP cinase/PKC / Abstract: Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for five days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-I8/syntaxin IA was decreased. Serine phosphorylation of SNAP-25, syntaxin IA, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactintreated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K+, 200 f.lMcarbachol and 1 f.lMPMA (pKC activator). The prolactin-induced increase in the formation of SNARE complex and syntaxin IA phosphorylation was inhibited by PD098059, a blocker of the MAPK pathway. These findings indicate that prolactin primes pancreatic B-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery, probably via the MAPKlPKC pathway / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Cellular communication requires the transport of chemical messengers between intracellular compartments and from cell to cell. The regulated exocytosis of a secretory vesicle at the plasma membrane involves the merger of two bilayers, with markedly different lipid composition, within a millisecond time scale. The spatial and temporal control of the protein and lipid complement at these fusion sites is essential. A highly conserved family of proteins are known to drive this fusion event; SNAP-25 and syntaxin-1 (t-SNAREs) associate at the plasma membrane in a 1:1 stoichiometry to provide a binding site for the vesicle-membrane protein synaptobrevin (v-SNARE). The formation of this complex and subsequent fusion requires accessory proteins for efficient calcium-triggered exocytosis; which of these proteins facilitate the initial attachment of vesicle to the plasma membrane prior to fusion is still under debate. Specific sites for vesicle fusion have been proposed and the organisation of lipids and proteins at these fusion sites has been extensively investigated with limited spatial and temporal resolution; however the presence of raft-forming lipids at these sites as well as the arrangement of SNARE proteins at the molecular level is still under contention. The data presented within this thesis aims to elucidate the protein and lipid environment at the fusion site using super-resolution microscopy and advanced vesicle tracking. Under diffraction-limited microscopy the t-SNAREs are visualised as 200 nm homogenous clusters; however I have used single molecule localisation microscopy to reveal a more complex heterogeneous molecular arrangement. Quantification of lipid order exclusively at the plasma membrane provided insight into the influence of cholesterol-induced lipid arrangement on SNAP-25 localisation. In addition the t-SNARE interaction was investigated using TCSPC-FLIM identifying two lipid-order-dependent conformations in distinct clusters at the plasma membrane. Extensive vesicle tracking at optimum sampling rates demonstrated the ‘sampling’ behaviour of LDCVs and allowed characterisation of vesicle fusion sites. In summary I find that vesicles exhibit preference for residence and probably fusion at regions of plasma membrane with a low t-SNARE density; these proteins appear to exert control over exocytosis by adopting alternative conformations that are under cholesterol-induced regulation.

No description available.

Tomosyn is a protein belonging to the Lgl family and conserved across the animal and plant kingdom. Tomosyn is composed of N-terminal domain containing WD40 motif and Cterminal domain, where the R-SNARE motif is located at the end of the C-terminal domain. This motif is classified as homologous to R-SNARE motif of synaptobrevine, which is a protein located on the surface of the vesicles and participating in the formation of SNARE complex and subsequent fusion of the vesicles with the plasma membrane. Thus the role of this tomosyn is mainly the regulation of exocytosis. Apart from the animal tomosyn, its yeast homolog Sro7/Sro77 is also examined, however during the evolution it has lost its R-SNARE motif and therefore the plant tomosyns remain unexplored. The aim of this diploma thesis was to characterize both of the plant tomosyns: AtTYN1 and AtTYN2 in Arabidopsis thaliana plant. The experimental thesis included the bioinformatic analysis, the DNA construct creation, a search for interactors by yeast two-hybrid system and monitoring the localization using the confocal microscope. The bioinformatic analysis results, including the creation of phylogenetic tree, not only revealed the conservation of tomosyns across the different classes, but also the division of both tomosyns into different clusters. There was...

Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

No description available.

No description available.

Harvest data are typically used to evaluate mesocarnivore population dynamics in many states, including Kentucky. While relatively easy to collect, these data are subject to reporting biases, and inferences about population trends can often only be made at coarse spatial scales. Gray fox (Urocyon cinereoargenteus), bobcat (Lynx rufus), and coyote (Canis latrans) populations in Kentucky are managed primarily through harvest data used to establish future harvest quotas. Increasingly, noninvasive survey methods have been used to characterize a number of population parameters for a variety of species; however, successful use of these methods is often site-specific. We assessed the efficacy and cost-effectiveness of two noninvasive survey methods, scat detection dogs and rub-pad hair snares, for surveying mesocarnivore species at two sites in the mixed-mesophytic forest of northeastern Kentucky. We sampled 100 hair snares covering approximately 100km2 and 27 transects covering approximately 27km2 from which 7 hair samples and 261 scat samples were collected respectively. Hair snares cost $397/sample at 6.4 hours/day, while scat detection dogs cost $47/sample at 4.9 hours/day. Genetic methods were used to identify biological samples to species and individual. Our findings should prove useful to state wildlife managers in comparatively evaluating methods for future mesocarnivore monitoring.