AbstractsBiology & Animal Science

Characterizing and quantifying membrane order of polarized epithelial cells in zebrafish larvae

by Ahmed Abu Siniyeh

Institution: University of New South Wales
Department: Centre for Vascular Research
Year: 2014
Keywords: Zebrafish; Membrane order; Polarity proteins; Lipid rafts proteins
Record ID: 1059700
Full text PDF: http://handle.unsw.edu.au/1959.4/53724


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,…