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

Abstract

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.