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.

<p>Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion <italic>in vivo</italic>. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia. </p><p>To identify the developmental requirements for <italic>cftr</italic> function, we generated <italic>cftr</italic> mutant zebrafish using transcription activator like effector nucleases (TALENs). In <italic>cftr</italic> mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in <italic>cftr</italic> mutants, which prevents directional fluid flow necessary for KV function. To examine <italic>cftr</italic> expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the <italic>cftr</italic> promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for <italic>cftr</italic> during KV morphogenesis and function in the zebrafish. </p><p>In the zebrafish pancreas, we found that loss of <italic>cftr</italic> function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of <italic>cftr</italic> BAC expression in the adult and larval zebrafish pancreata revealed that <italic>cftr</italic> is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult <italic>cftr</italic> mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that <italic>cftr</italic> is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and <italic>cftr</italic> mutant larvae. These results indicate that <italic>cftr</italic>-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of <italic>cftr</italic> function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.</p> / Dissertation

Earth’s crust and primordial ocean formed more than 4 billion years ago and life is believed to have originated on earth at least 3.6 billion years ago. This suggests that primitive cellular life must have evolved from non-living matter during that period of several hundred million years. To study the transition from chemistry to biology, a simple vesicular system called a protocell is an ideal model that is self-organized and contains informational or metabolic materials. This thesis starts by exploring the replication of a model genetic material under plausible prebiotic conditions. The non-enzymatic copying of RNA was found to be catalyzed by Fe2+, which used to be abundant in aqueous environments on the early anoxic earth. Fe2+ was found to be a better catalyst of non-enzymatic RNA copying and ligation in slightly acidic to neutral pH conditions than Mg2+, the divalent cation used to catalyze these reactions in previous studies. This finding suggests that ferrous iron could have facilitated the replication and evolution of RNA on the prebiotic earth. To gain a better understanding of the properties of protocell membranes, the impact of membrane composition and multi-bilayer structure on non-enzymatic and enzymatic biochemical reactions was studied. A fatty acid/phospholipid hybrid membrane system was proposed as a potential intermediate state in protocellular evolution. This membrane composition was investigated for its stability and permeability, two fundamental features of functional protocells. The system proved stable in the presence of divalent cations and retained permeability to small building block molecule. Vesicles with this composition were shown to host faster non-enzymatic RNA copying, and to enable enzymatic protein synthesis. To study the effects of multi-lamellarity, giant multilamellar vesicles (GMVs) were prepared by an extrusion-dialysis method. Compared with small unilamellar vesicles (SUVs), GMVs show slightly better ability to retain encapsulated RNA, while maintaining good permeability for small charged molecules. The multilamellar structure also promotes non-enzymatic RNA copying, providing preliminary evidence that membranes could also mediate catalytic functions as well as acting as a compartment. / 2020-07-02T00:00:00Z

About 5% to 10% of epileptic patients suffer from Juvenile Myoclonic Epilepsy (JME), which is characterized by spasms of the arms, ataxia (uncoordinated movements), and general tonic-clonic seizures. In a recent study, a group of patients with myoclonic epilepsy was found to harbor mutations in the PRICKLE1 and PRICKLE2 genes. This suggested that PRICKLE genes might be linked to epilepsy, and given that PRICKLE is highly evolutionarily conserved (including in fruit flies), we decided to use Drosophila in order to determine, first, whether flies with prickle mutations were seizure-prone, and if so, to then use the powerful genetic tools of Drosophila to elucidate the underlying mechanism of the prickle-associated epilepsy. In this work, we show that mutation of the pksple isoform (one of the two adult prickle isoforms in flies) lowers the seizure threshold in the mutant flies (resulting in seizure activity), while mutation of the other adult isoform, pkpk, had no effect. This was demonstrated through both behavioral assays (where the pksple mutant flies showed a reduction in recovery of climbing behavior after being subjected to mechanical stimulation while the pkpk mutant flies did not) as well as electrophysiological analysis (where pksple mutants were shown to be hyperexcitable after electrical stimulation, while the pkpk allele showed no change in spiking activity). We demonstrated that the underlying mechanism of the hyperexcitability seen in the pksple flies was due to enhanced anterograde transport on microtubule (MT) tracks in neurons, the main route for transport in neurons, which could be suppressed by reducing the dose of either of two Kinesin motor proteins, the motors involved in anterograde transport in neurons. On the other hand, the pkpk mutants showed the reverse effect, exhibiting a significant reduction in vesicle transport dynamics. We showed that microtubule polarity could be partially reversed by tipping the balance of the pk isoforms similar to what is seen in the pkpk mutants (such that a large percentage of MTs now had their plus ends oriented towards the cell body, which is extremely rare in axons), suggesting that the vesicle transport defects seen in the pkpk mutants might be due to mixed polarity of MTs. Next, we showed that the seizure-prone pksple mutants, but not the pkpk mutants, exhibited a myoclonic form of epilepsy, as well as abnormal walking patterns and uncoordinated movements, paralleling the ataxia phenotype seen in the epileptic patients with PRICKLE mutations. These data suggest that the primary aspects of the epilepsy-ataxia syndrome seen in patients with PRICKLE mutations are recapitulated in flies, which underscores the utility of using the fruit fly genetic system to model this disorder. Finally, our preliminary results suggest that the pk alleles have different effects on neuronal morphology due to changes in sizes of terminal boutons at the neuromuscular junction (NMJ) in larvae. These data suggest that pk is having a direct effect on synaptic formation and likely function. In conclusion, by using our Drosophila model system, we were able to link prickle mutations to epilepsy as well as identify the cellular mechanism of the prickle-associated epilepsy, a novel epilepsy mechanism previously associated with neurodegeneration. To our knowledge, this is the first example of a gene that, when mutated, will cause seizures in flies, zebrafish, mice, and humans, indicating that the role of prickle in controlling seizure activity is remarkably conserved in animals. Significantly, since about one third of patients with epilepsy do not respond to current AEDs, our fly model and the techniques we have developed will enable us to conduct drug screens for testing potential chemical compounds as new AEDs.

Background: Escherichia coli (E.coli) O86a:K61 belongs to the Enteropathogenic E. coli (EPEC) group of pathogens. Acute gastroenteritis affects 2-4 billion people annually and EPEC is associated with 10-40% of hospitalized diarrhea cases globally. Coagulation Factor (F) V circulates as an inactive procofactor (Mr 330kDa) which upon thrombin activation to the active cofactor, FVa, functions in prothombinase to accelerate prothrombin to thrombin conversion by 300,000-fold. The ability of E.coli O86a:K61 to cause intestinal hemorrhage is of interest because previous research demonstrated that during E.coli O86a:K61 sepsis in baboons, a dose-dependent inactivation of FV was observed as the bacterial dose increased. These results suggested a secreted E.coli protease may have mediated this effect on FV. This research has focused on the purification, identification, and characterization of a secreted E. coli O86a:K61 protease that inactivates FV. The final partially-purified protease inactivated FV to a 250kDa product by immunoblotting, and possessed a 900-fold increase in specific activity versus FV in human plasma compared to the culture supernatant. At least 3 proteins were observed upon SDS-PAGE. Proteolytic inactivation of FV was activated by up to 500-fold with β-mercaptoethanol and 2-fold with 1M urea. The protease was heat stable retaining all of its activity versus FV after 1h at 70°C or 80°C, and partial activity (50%) at 95°C. Proteolysis of FV was blocked by 90% with alpha-1-protease inhibitor; however, the protease was resistant to 1.5 mM PMSF, and unaffected by E64, or iodoacetamide. FV is a major regulator of the coagulation process and its inactivation by the secreted E.coli protease would be expected to result in a net bleeding tendency which may contribute to the mucosal hemorrhage observed in humans with associated hemorrhagic colitis. Proteolytic inactivation of FV is predicted to result in decreased bacterial containment by host fibrin thereby increasing pathogen survival and growth. FV inactivation by the secreted E.coli protease may be part of a novel pathogenic virulence mechanism that deregulates the blood coagulation process to enhance bacterial infectivity and transmission. / UOIT

Naja naja atra Cardiotoxins (CTXs), basic polypeptides of 60 amino acid residues adopt a three-fingered loop-folding topology and show cytotoxicity for human tissues in targeting cell membrane. Despite having highly similar sequence, the six CTX isoforms also display different cytotoxic potencies and hemolytic activities. The goal of these studies is to explore the mechanical processes that involved in membrane-damaging activities of CTXs on vesicles composed of different cell membrane components, and to delineate the events that lead to different biological activities of CTXs. The studies were performed by estimating the color transformation of phospholipid/polydiacetylene vesicles and the fluorescence enhancement of fluorescein-labeled phospholipid/protein or fluorescein released from vesicles. It was found that vesicles consisted of unsaturated phospholipids improve membrane-damaging activity of CTXs and adopt a vital membrane-bound conformation of CTXs. In contract, the characteristic of vesicles consisted of saturated phospholipids was against CTXs adopting an essential membrane-damaging structure. It was also found that not only electrostatic force but also hydrophobic force were involved in the interaction between CTXs and membrane. Comparing with phosphatidylcholine-only vesicles, CTXs displayed higher membrane-damaging activity for the sphingomyelin-containing vesicles, and the loop2 region of CTXs play a crucial role for the membrane-damaging activity of sphingomyelin-containing vesicles. Besides, the CTX3 and CTX5 would interact with the H-antigen of blood group O red blood cells, but only the binding of CTX3 with H-antigen reduce its membrane-damaging activity for red blood cells membrane. Moreover, the fusogenicity of CTXs is responsible for the membrane-damaging activity of CTXs toward bacterial membrane-mimicking vesicles. The cardiolipin have the potency to improve the fusogenicity of CTX3, which induced the bactericidal activity toward the cardiolipin-containing bacterium.

No description available.

T cells play pivotal roles in the immune system and focused secretion of either cytokines or cytotoxic molecules toward its target is crucial for T cell functions. This directional secretion involves two critical steps: the movement of the microtubule organizing center (MTOC) up to the cell-cell contact site and the directed movement of secretory vesicles towards the MTOC. The minus end-directed microtubule motor protein dynein was previously shown in our studies and those of others to accumulate and anchor at the contact site where it then draws the MTOC up to the contact site. A variety of studies led to the suggestion that there were two functionally different pools of dynein in Jurkat cells, one a ring-like structure that pulled the MTOC to the contact site and the other one uniquely corresponding to the distribution of dynactin. This led to the hypothesis that the second pool of dynein drove vesicle transport. To address this possibility, we used siRNA to deplete the cell of dynactin. These studies showed that almost complete knockdown of dynactin (p150[superscript Glued]) had little effect on MTOC translocation but it also had little effect on a panel of Golgi vesicle markers, whose movement the literature suggested was dynein dependent. As an alternative, a Jurkat cell line expressing fluorescent CTLA4, a known marker for the secretory lysosomes was generated. CTLA4 accumulated at the contact site when Jurkat cells made contact with synthetic target cells. When we repeated the p150[superscript Glued] knockdown in these cells, we found that vesicle transport was blocked, whereas MTOC polarization remained normal. These studies suggest that dynein serves critical roles in both aspects of T cell effector function, the movement of the MTOC up to the cell-cell contact site and the movement of a special class of secretory vesicles up to the MTOC. By the combined processes of MTOC translocation and the minus end-directed movement of vesicles, T cells make it so that a concentrated pool of secretory vesicles are aimed to secrete locally only towards target cells. This ensures that the antigen-specificity of T cell activation is followed by a localized response aimed at the intended target cell. / text

The intention of this study is to investigate the evolution and transition of lyotropic lamellar phase and the formation of multi-lamellar vesicles (MLVs) under shear flow, since the shear technology can be used to produce well defined multi-lamellar vesicles which are useful for encapsulating drugs in medical or research fields. The system was designed to stabilize and track one single multi-lamellar vesicle, which is being sheared under Couette shear flow between two co-rotational disks, by using polarizing microscope and a LabView program. For the whole system, most parts of the hardware instrument and all the software programs were originally designed and homemade, which makes this a unique undertaking. Eighty percent of the time was spent on designing, assembling, testing and improving the hardware instrument and software programs to make sure the system can achieve our aim as accurately as possible. The lyotropic lamellar phase sample is made of pentanol, dodecane, SDS and water. Nine different concentrations from 16% to 32% of SDS+Water were explored under five different shear rates from 3.3 to 13.2 . Sodium dodecyl sulfate (SDS) is a kind of surfactant which has an amphiphilic molecular structure, and a certain liquid crystal structure (such as a lamellar phase) will be formed when it is dissolved in a water/oil mixture solvent. It is a great achievement that one single multi-lamellar vesicle is able to be followed for over 20 minutes under shear, and it is found that the multi-lamellar vesicle does not exhibit any obvious changes with time once it was already formed. Three different structural regions were found for the dilute lamellar phase while evolving to the multi-lamellar vesicle orientation state under shear. However, only two regions were found for the lamellar phase with higher concentrations under low shear rate since the lamellar phase will not reach to the multi-lamellar vesicle state. Besides, on the basis of the results of these experiments, it can be concluded that either higher shear rate or higher concentration of SDS+Water will hasten the formation of multi-lamellar vesicles. For the transition time of reaching a uniform multi-lamellar vesicle orientation state, it can be reduced by increasing shear rate. In addition, the results show that the transition time is decreasing more slowly for high concentrated lamellar phases than dilute lamellar phases with increasing the shear rate.

No description available.