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.