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The membranes of olfactory receptor neurons (ORNs) need to aid the detection of odourants and the generation of appropriate signals that are transmitted to the central nervous system. Odourant receptors and their signalling machinery are located in thin membrane protrusions called cilia. Ciliary membranes must withstand the harsh environment of the nasal cavity while maintaining a fluid lipid bilayer that enables regulation of the olfactory signalling cascade and generation of the olfactory response. The organisation of ORN ciliary membranes and the functional consequences of this organisation are poorly understood. To quantitatively assess the organisation of plasma membranes of functionally and anatomically different membrane domains of ORN, environmentally sensitive membrane dyes, Laurdan and di-4-ANEPPDHQ in conjunction with two-photon and confocal laser-scanning microscopy were employed. The emission spectra of these fluorophores correlate with the degree of membrane hydration that can be quantified as a Generalised Polarisation (GP). In this study it was found, that the membrane order (MO) in different membrane domains on the surface of the ORNs had similar GP values, in isolated amphibian ORNs as well as in mammalian ORNs, which were still embedded within olfactory epithelium (OE). However, when membrane lipid content was altered or when odourants were present, MO exhibited domain-specific changes. Also it was demonstrated, that odourants from chemically different groups had different and domain-specific effect in OE. To better understand the complex relationship between olfactory signalling proteins, odourants and MO, a heterologous system was employed. The A2 subunit of CNG channel and isoform B of CaCC, ANO2, was transiently expressed in HEK293T/17 cells. The lipid composition of these cells was manipulated and cells were exposed to different concentrations of the odourant 1-heptanol. The findings confirmed that odourant can directly affect MO. Effects of the odourant were not only dose-dependent, but also dependent on membrane lipid composition. Both of the investigated olfactory signalling proteins demonstrated the ability to regulate MO when membrane lipid content was altered and/or odourant was present. Hence, the role of the signalling proteins in olfaction may not be limited to signalling processes but may also be extended to the maintenance of membrane organisation.

The composition and structure of plasma membranes is critical for many cell functions. The plasma membrane of polarized epithelial cells can be divided into two compartments, the apical and basolateral membrane that differ in compositions and function. In particular, it has been proposed that the apical membrane is enriched in lipid rafts. Lipid rafts are defined as small lipid domains that are enriched in cholesterol and sphingolipids. Thus a biophysical property of raft membranes is that they are more ordered than non-raft membranes. The apical-basolateral polarity in polarized epithelial cells is maintained by polarity proteins. Polarity proteins typically localized to either apical or basolateral membranes and organize intracellular trafficking to either membrane compartment. It is currently unknown whether and how membrane organization and polarity networks are linked in polarized epithelial cells. The purpose of the research presented in this PhD thesis was to investigate the relationship membrane order and polarity protein localization. A better understanding of the cell membrane and its biophysical properties can be achieved by visualizing and analyzing membranes in whole vertebrate organisms rather than imaging cells in tissue culture systems. This is because in vivo, the cellular organization within organs is maintained, which is not just critical for the polarization of epithelial cells but also likely to affect the physical-chemical properties of cell membranes. Here, zebrafish was used as a model organism because its transparency enables high-resolution fluorescence imaging. Zebrafish has become a popular animal model because embryos can be genetically manipulated to study the molecular basis for disease including diseases in which epithelial cell polarization is a key factor. In this study, the transparency of zebrafish was exploited to study the relationship between membrane order and the localization of polarity proteins in epithelial cells in three different tissues: gut, kidney, and liver. Using the membrane dye Laurdan and multi-photon microscopy, membrane order of polarized epithelial cells in the gut, kidney, and liver were quantified at different development stages. Laurdan incorporates itself into cell membranes parallel to the hydrophobic tails of phospholipids. The probe displays spectral sensitivity to the polarity of its environment, with a ∼50-nm red shift of its emission maximum in polar versus nonpolar environments. This shift in emission profile allows a quantitative assessment of membrane order by calculating a ratiometric measurement of the fluorescence intensity recorded in two spectral channels, known as a generalized polarization (GP) value. A change in membrane order in epithelial cells was observed during development with particularly high membrane order recorded at 6 days post fertilization (dpf) for all three tissues. Apical membranes were significantly more ordered than the basolateral membranes, and basolateral membranes were more ordered than intracellular membranes in gut, kidney, and liver at 3-11 pdf. Manipulation of 6 dpf larvae with either 7-ketocholesterol (7KC) or cholesterol- methyl-β-cyclodextrin complexes (cholesterol-mβCD) or methyl-β-cyclodextrin (mβCD) significantly decreased membrane order in the apical, basolateral and intracellular membranes in gut, kidney, and liver epithelial cells. When 4 dpf larvae were treated with 7KC, cholesterol-mβCD or mβCD, the membrane order of apical, basolateral and intracellular membranes was also significantly decreased but recovered to almost normal levels when 4 dpf sterol-manipulated larvae were grown to 6 dpf. Tissue organization was largely unaffected by the sterol manipulations but apical targeting of atypical protein kinase C (aPKC) was reduced in sterol-manipulated 4 pdf larvae that also recovered after a further two days of development. Therefore, a strong correlation between the high membrane order and apical localization of aPKC was observed under conditions where membrane order was acutely decreased and post recovery. To investigate whether polarity and membrane proteins influence membrane order in polarized epithelial cells, morpholinos (MO) were used to knockdown the expression of the polarity proteins Par3 (par3 MO) and Crb3a (crb3a MO), and the lipid raft proteins, Flotillin-1a (flot-1a MO) and Flotillin-2 (flot-2a MO). A significant decrease in membrane order was found in the apical, basolateral and intracellular membranes in epithelial cells in the gut, kidney, and liver of par3, crb3a, flot-1a, and flot-2a morphants larvae at 4 dpf compared to control. In contrast, a decrease in apical localization of aPKCs was only observed in epithelial cells of par3 and crb3a morphants larvae but not in flot-1a and flot-2a morphants larvae. In conclusion, the data suggest that membrane order and polarity protein networks are mutually dependent on each other in polarized epithelial cells of the gut, kidney, and liver of intact zebrafish larvae. Decrease in membrane order resulted in a diminished apical localization of the polarity protein aPKC and reduced expression of the polarity proteins Par3 or Crb3a decreased membrane order. This strongly suggests that polarity networks and membrane organization are not controlled by two distinct cellular processes and networks but are part of the same biology. It is possible that the delivery of lipids to apical and basolateral membranes by polarity proteins maintains the differences in order in these membranes and that membrane order is a trafficking ‘signature’ for apical sorting. Hence, the work presented here is only the first step towards a complete picture of the functional relationship between polarity proteins and membrane organization. The multidisciplinary approach taken here may inspire future studies to better integrate the biology of membranes with the biology of polarized trafficking.

As the T cell receptor and many of the associated signalling molecules are embedded within the plasma membrane, it has been hypothesised that plasma membrane lipids, and associated lipid ‘ordered’ membrane domains, play a role in regulating T cell signalling. The first aim of this thesis was to examine in mice whether dietary lipids influence the lipid composition of T cells and thus membrane order, and how this correlates to in vivo and ex vivo T cell responses. Chapters 3 and 4 describe both acute and long-term dietary studies, which involved feeding C57BL/6J and LDLR-/- mice a high-fat or high-cholesterol diet. A contact hypersensitivity (CHS) reaction was then induced using 1-fluoro-2,4-dinitrobenzene and the CD4+ and CD8+ T cell responses examined in the draining lymph nodes by flow cytometry. The in vivo T cell responses were correlated to the cellular lipid content of splenic T cells using mass spectrometry, and membrane order measured with Laurdan microscopy. Finally, the activation response of ex vivo stimulated splenic T cells was assessed with qRT-PCR. Both the C57BL/6J and LDLR-/- dietary studies illustrated that long-term high-fat or high-cholesterol feeding significantly increased both CD4+ and CD8+ T cell proliferation, and central-memory CD4+ T cell production, within the draining lymph nodes following CHS. This was associated with changes in cellular phospholipids, fatty acids and cholesterol, as well as alterations in membrane order at the T cell activation site, and reduced IL-2 mRNA production. The last results chapter of this thesis, chapter 5, focused directly on how membrane order impacts on T cell function in vivo. Using an adoptive transfer system, CD4+ T cells from OT-II transgenic mice were treated with the oxysterol 7-ketocholesterol, which perturbs membrane condensation, or a cholesterol control, before transfer into B6.SJL recipient mice. I then challenged the recipients with ovalbumin323–339 to measure antigen-specific CD4+ T cell responses by flow cytometry, which were again correlated the sterol content of ex vivo manipulated cells. Our results were inconsistent but suggest that both 7KC and cholesterol treatment cause a reduction in OVA-specific CD4+ T cell proliferation within the draining lymph nodes of recipient mice.