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

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

The Association between Adiponectin Single Nucleotide Polymorphisms and Side Effects of Isotretinoin in Acne Patients

Background. Acne is a common condition of pilosebaceous follicle especially among young. Clinically, the most used medication in the treatment of moderate to severe acne is oral isotretinoin. However, interindividual variability in therapeutic response to isotretinoin and many side effects such as musculoskeletal pain, headache, and alteration in lipid profile can be seen with this treatment. Aim. In this study, the effect of genetic polymorphisms, rs2241766 and rs1501299, of the adiponectin gene was investigated in relation to the side effects of isotretinoin-treated young adult acne patients (n = 230). Methods. Several biochemical parameters were measured at baseline and after treatments with isotretinoin. The ADIPOQ gene SNPs, rs2241766 and rs1501299, were genotyped in 230 patients. Results. Alterations in lipid profile with a significant increase of ALT (P=0.007) were detected after isotretinoin treatment. Moreover, percentage change in HDL following isotretinoin treatment was significantly associated with rs1501299 (P=0.008). On the other hand, no associations between examined SNPs and side effects of isotretinoin and other lipid parameters (total cholesterol, LDL, and triglycerides) or liver function enzymes (ALT and AST) were detected. Conclusions. Current findings showed that rs1501299 of the ADIPOQ gene might be associated with changes in HDL level in acne patients following treatment with isotretinoin

Characterization of a new series of fluorescent probes for imaging membrane order

Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology

Attitude towards donation of the excised foreskin after circumcision surgery for research: A study from Madinah, Saudi Arabia.

Studies have shown the possibility of using the part of the foreskin removed after circumcision in the field of scientific and therapeutic research. Donations of tissues and organs are always associated with ethical challenges posed by bioethicists and societies to ensure the appropriate use of these tissues/organs. The purpose of this study was to understand the attitudes and awareness of parents/guardians regarding donation of excised foreskin to research and medical use. The study was based on a questionnaire and included 133 parents/guardians who visited Uhud Children's Hospital in Madinah, Saudi Arabia for newborn male circumcision. The results showed a high willingness (61.7%) to donate the extracted foreskin to research. The willingness to donate the extracted foreskin to research associated with undergraduate degree (P = 0.018), male sex (P = 0.011), high income (P = 0.029), and participation in previous research studies (P = 0.002). About 41.8% were convinced that written informed consent should be obtained before circumcision surgery, 38.1% (n = 51) were convinced that written informed consent should be taken after surgery, while the remaining 19.4% reported that the timing of written informed consent is unimportant. Finally, fear of excision of excess tissue (74.5%), lack of confidence in the research (68.6%), and potential for commercial use (64.7%) were the main barriers to unwillingness to donate the excised foreskin for research. In conclusion, a reasonable portion of Saudis agreed to donate their foreskin for research purposes. There is an urgent need to enhance awareness and attitudes towards tissue donation for research and therapeutic use

Analysis of live Zebrafish embryos.

<p>Top: Histograms of the GP values obtained from GP images of live Zebrafish embryos stained with PY3304 (A), PY3174 (B), PY3184 (C). Histograms obtained from ROIs for plasma membrane (red line) and intracellular membranes (black) were normalized to the total number of pixels. Bottom: GP images are in false color and run over the range indicated by the color bars to indicate a higher degree of membrane order (predominately colored red) in the plasma membrane compared to intracellular membranes (predominately colored green). Embryos were stained with PY3304 (left), PY3174 (middle), PY3184 (right) excited by multi-photon excitation at 1040 nm for PY3304, 900 nm for PY 3174, 1000 nm for PY 3184. Scale bars  = 10 µm.</p

Analysis of Giant Unilamellar Vesicles.

<p>Top: Histograms of the GP values obtained from 2-channel confocal images of artificial bilayers in liquid ordered phase (red line) and liquid disordered phase (black line) obtained with PY3304 (A), PY3174 (B), PY3184 (C). Histograms are normalized to the total number of pixels. Bottom: GP images of 1∶1∶1 DOPC:PSM:cholesterol GUVs showing coexistence of ordered and disordered phase membranes stained with the three probes and imaged by 2-channel confocal microscopy. Scale bar  = 5 μm.</p

Analysis of live HeLa cells.

<p>Left: GP images of live HeLa cells stained with PY3304 (A), PY3174 (B), PY3184 (C). GP images are in false color and run over the range indicated by the color bars to indicate a higher degree of membrane order (predominately colored red) in the plasma membrane compared to intracellular membranes (predominately colored green). Scale bars 10 µm. Middle: Histograms of the GP values obtained from GP images of live HeLa cells stained with PY3304 (A), PY3174 (B), PY3184 (C). Histograms obtained from ROIs for plasma membrane (red line) and intracellular membranes (black) were normalized to the total number of pixels. Right: Staining profile of Live HeLa cells. HeLa cells were incubated with PY3304 (A), PY3174 (B) and PY3184 (C) for 30 min. Confocal intensity images were acquired at 30 s, 10 min, 20 min, and 30 min after dyes were added. Images show complete staining of all cellular membranes after 30 min. Scale bars 50 µm.</p

Excitation and emission spectra of artificial bilayers.

<p>The wavelength bands for 2-channel acquisitions are indicated by shaded boxes. Dashed line corresponds to liquid ordered phase and continuous line corresponds to liquid disordered phase. The solid vertical line corresponds to the excitation wavelength used for microscopy experiments.</p

Analysis of FLIM images.

<p>Left: Fluorescence decay histograms acquired from artificial membranes stained with PY3304 (A), PY3174 (B) and PY3184 (C) showing longer lifetimes in ordered membranes (red) than disordered membranes (black). Middle: Plots of residuals from fitting fluorescence decay histograms. (D-F) Right: Fluorescence lifetime images of live HeLa cells stained with PY3304 (D), PY3174 (E), PY3184 (F). Images show an increased order at the plasma membrane in agreement with the spectral measurements and previously published results. Scale bar  = 10 μm.</p