The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.

Renin is the initiator and rate-limiting factor of the renin-angiotensin system, a major mechanism of blood pressure regulation. The synthesis and secretion of this active circulatory enzyme is confined exclusively to the dense core granules of kidney juxtaglomerular (JG) cells where its precursor prorenin is packaged, cleaved to the active form and stored for release on a regulated pathway. Given its importance, surprisingly little is known about this process, in part due to the difficulty in culturing primary JG cells in vitro and the lack of reliable cell lines. The initial aim of the current work was to investigate renin-containing granule dynamics in live JG cells. To achieve this, I attempted to derive novel cell lines from triple transgenic mouse models comprising immortalised granulated or non-granulated JG cells. Due to the nature of JG cells in culture, the use of these cell lines to investigate granulation was not feasible; therefore the culture of primary JG cell culture was modified and enhanced to visualise granule dynamics in live, primary JG cells for the first time. By isolating cells using a Percoll gradient and plating them on fibronectin-coated dishes, rapid and full adhesion of JG cells was achieved, as well as prolonged expression of renin from 3 days to up to 8 days post-isolation. Using this protocol to isolate JG cells from RenGFP renin reporter mice and identifying granules using the acidotropic fluorophore Lysotracker, granule dynamics were investigated in primary JG cells. High resolution, rapid image acquisition was performed using widefield and total internal reflection microscopy, showing that dense core granules respond dynamically to the β-adrenergic agonist isoproterenol, a known renin secretory stimulus. Two different pools of granules of varying granule diameters and dynamic parameters were identified optically, suggesting that separate granule pools were being identified. Mice null for the Ren-1d gene lack renin storage granules in their JG cells, however granulation was restored in Ren1d-null mice carrying a transgene encompassing the human renin (hRen) locus. Therefore in order to investigate the relationship between renin expression and the amount of granulation in JG cells, mice expressing human renin were used. To dissect the granulation phenotype in detail, 2D electron micrographs were taken of JG cells, which were immunogold stained to confirm renin content, and reconstructed in 3D. Female hRen mice showed a significantly higher volume of granulation and an increased granule number compared to males, a finding consistent with the sexually dimorphic expression of the transgene, supporting the hypothesis that granulation in JG cells is dependent on the level of renin expression. The macula densa (MD) is a critical sensor of flow and salt content in the blood; through extensive tubulo-vascular crosstalk known as tubuloglomerular feedback (TGF), it releases key signalling factors stimulating and inhibiting renin synthesis and secretion from JG cells. Ren-1d-/- mice showed a hypercellular and columnar MD plaque, which was not restored by the introduction of the hRen transgene, indicating that TGF may be impaired in these mice. Using an isolated, perfused juxtaglomerular apparatus model it was shown that high salt- and increased flow-induced TGF functioned effectively in Ren1d-/- and huRen+/-Ren1d-/- mice, although animals on a Ren1d-/- background showed decreased sensitivity of glomerular tuft contraction and abnormal calcium signalling within macula densa cells. In conclusion, an appropriate in vitro model was developed for investigating granule dynamics in JG cells, using which granule motion was visualised and quantified for the first time in these cells. Although JG cell granulation is required for normal MD morphology, it was shown to not affect JGA function.

Actin cytoskeleton reorganization plays essential roles in many cellular processes such as cell structure maintenance, cell motility, and force generation. Cytoskeletal drugs are small molecules that act on cytoskeletal components by either stabilizing or destabilizing them. Swinholide A is an actin-binding drug derived from the marine sponge. Swinholide A binds actin dimers as well as severs filaments. The main objective of this project is to determine how Swinholide A modulates actin filament assembly dynamics in the presence of macromolecular crowding. We utilize total internal reflection fluorescence (TIRF) microscopy imaging to directly visualize Swinholide A-mediated actin filament disassembly and severing. Filament disassembly and severing are evaluated by calculating actin filament lengths and length distribution controlled by Swinholide A. This study helps us better understand the fundamental mechanism by which Swinholide A affects actin assembly and disassembly dynamics. Further studies will allow for investigating new methods of treatment for a range of different diseases that have pathogenetically high levels of filamentous actin, such as cystic fibrosis, as well as a drug to combat the explosive expansion of cancers.

Understanding the mechanisms by which proteins act on membrane surfaces is fundamental if we are to exploit their capabilities or halt the progression of the diseases they are associated with. Arguably, the best way to study these interactions is by using techniques that can obtain molecular-scale information, in real time and under physiologically relevant conditions. Studying supported lipid bilayer systems with high spatial resolution tools, such as atomic force microscopy (AFM), and high temporal resolution techniques, such as polarized total internal reflection fluorescence microscopy (pTIRFM), allows us to meet these requirements [1]. The goal of this project is to use methods that are currently available and further their applications and capabilities to provide insight into the mechanisms by which amyloidogenic and antimicrobial peptides act on membranes.

Understanding the mechanisms by which proteins act on membrane surfaces is fundamental if we are to exploit their capabilities or halt the progression of the diseases they are associated with. Arguably, the best way to study these interactions is by using techniques that can obtain molecular-scale information, in real time and under physiologically relevant conditions. Studying supported lipid bilayer systems with high spatial resolution tools, such as atomic force microscopy (AFM), and high temporal resolution techniques, such as polarized total internal reflection fluorescence microscopy (pTIRFM), allows us to meet these requirements [1]. The goal of this project is to use methods that are currently available and further their applications and capabilities to provide insight into the mechanisms by which amyloidogenic and antimicrobial peptides act on membranes.

Thesis advisor: Mary F. Roberts / Thesis advisor: Steven D. Bruner / The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from <italic>Bacillus thuringiensis</italic> is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling <super>31</super>P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

This research presents a detection scheme for analyzing the temperature distribution produced by the Joule heating effect nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system¡Xtotal internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the inner channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective-type TIRFM, microscope cover glass are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM makes the intensity information obtained is not sensitive to the channel depth variation which ususally biases the measured results while using conventional epi-fluorescence microscope (Epi-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within hundreds of nanometer of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. In order to investigate the temperature distribution produced by the Joule heating effect for electrokinetically driven microchips, this study not only measures the temperature on the microchannel wall by the proposed TIRFM but also measures the temperature inside the microchannel by an Epi-FM. In addition, this research presents a method to reduce the Joule heating effect and enhance the separation efficiency of DNA biosamples in a chip-based capillary electrophoresis (CE) system utilizing pulse DC electric fields. Since the average power consumption is reduced by the pulse electric fields, the Joule heating effect can be significantly reduced. Results indicate the proposed TIRFM method provides higher measurement sensitivity over the Epi-FM method. Significant temperature difference along the channel depth measured by TIRFM and Epi-FM is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the Joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a simulation model for precisely predicting the Joule heating effect in microfluidic chips. Moreover, in the research of reducing the Joule heating effect and enhancing the separation efficiency in a chip-based CE system utilizing pulse electric fields, the experimental and numerical investigations commence by separating a mixed sample comprising two fluoresceins with virtually identical physical properties. The separation level is approximately 2.1 times higher than that achieved using a conventional DC electric field. The performance of the proposed method is further evaluated by separating a DNA sample of Hae III digested £XX¡V174 ladder. Results indicate the separation level of the two neighboring peaks of 5a (271 bp) and 5b (281 bp) in the DNA ladder is as high as 120% which is difficult to be achieved using a conventional CE scheme. The improved separation performance is attributed to a lower Joule heating effect as a result of a lower average power input and the opportunity for heat dissipation during the zero-voltage stage of the pulse cycle. Overall, the results demonstrate a simple and low-cost technique for achieving a high separation performance in CE microchips.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

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