Oral Presentation 2014 International Biophysics Congress

Studying lateral organization in complex eukaryotic membrane extracts using Brewster angle microscopy (#168)

Mark Mahadeo 1 , Ola Czyz 1 , Simon Lam 1 , Jens Coorssen 2 , Vanina Zaremberg 1 , Elmar Prenner 1
  1. Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
  2. Molecular Physiology Department, and the Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia
Brewster Angle Microscopy (BAM) is an imaging technique used to characterize lateral organization in biologically relevant monolayers of lipids and lipid/protein mixtures. This technique utilizes light reflected from the monofilm and thus avoids the introduction of non-native labels and probes into the system. In the current work, BAM is used to study the domain organization in two eukaryotic systems. In the first instance Saccharomyces cerevisiae serves as a model organism to study the effects of the anti-tumour lipid drugs edelfosine, miltefosine and perifosine on the lateral organization of yeast membranes. While the mode of action is not fully understood, it is believed that these drugs accumulate preferentially in more rigid, so called liquid- ordered lipid domains. This leads to a breakdown of these membrane structures, causing a disruption of various cellular functions, including signal transduction, membrane trafficking and metabolism within the cell, finally causing death. Cells were treated with one of the 3 drugs and membranes were extracted using a modified Folch method. Results show a change in domain size, frequency and morphology due to treatment with edelfosine and miltefosine, but surprisingly not with perifosine. In the second system, the effect of cholesterol on the fusion of cortical vesicles isolated from oocytes of Strongylocentrotus purpuratus, was correlated to changes in domain organization observed by using BAM. In this work, depletion of cholesterol, shown to result in decreased vesicle fusion, was found to cause both an increase in domain size and decrease in frequency of domains within the extracted membranes.