4 research outputs found
Combining Reflectometry and Fluorescence Microscopy: An Assay for the Investigation of Leakage Processes across Lipid Membranes
The passage of solutes across a lipid
membrane plays a central
role in many cellular processes. However, the investigation of transport
processes remains a serious challenge in pharmaceutical research,
particularly the transport of uncharged cargo. While translocation
reactions of ions across cell membranes is commonly measured with
the patch-clamp, an equally powerful screening method for the transport
of uncharged compounds is still lacking. A combined setup for reflectometric
interference spectroscopy (RIfS) and fluorescence microscopy measurements
is presented that allows one to investigate the passive exchange of
uncharged compounds across a free-standing membrane. Pore-spanning
lipid membranes were prepared by spreading giant 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC) vesicles on porous anodic
aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments.
The time-resolved leakage of different dye molecules (pyranine and
crystal violet) as well as avidin through melittin induced membrane
pores and defects was investigated
Permeabilization Assay for Antimicrobial Peptides Based on Pore-Spanning Lipid Membranes on Nanoporous Alumina
Screening tools to study antimicrobial
peptides (AMPs) with the
aim to optimize therapeutic delivery vectors require automated and
parallelized sampling based on chip technology. Here, we present the
development of a chip-based assay that allows for the investigation
of the action of AMPs on planar lipid membranes in a time-resolved
manner by fluorescence readout. Anodic aluminum oxide (AAO) composed
of cylindrical pores with a diameter of 70 nm and a thickness of up
to 10 μm was used as a support to generate pore-spanning lipid
bilayers from giant unilamellar vesicle spreading, which resulted
in large continuous membrane patches sealing the pores. Because AAO
is optically transparent, fluid single lipid bilayers and the underlying
pore cavities can be readily observed by three-dimensional confocal
laser scanning microscopy (CLSM). To assay the membrane permeabilizing
activity of the AMPs, the translocation of the water-soluble dyes
into the AAO cavities and the fluorescence of the sulforhodamine 101
1,2-dihexadecanoyl-<i>sn</i>-glycero-3-phosphoethanol-l-amine
triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were
observed by CLSM in a time-resolved manner as a function of the AMP
concentration. The effect of two different AMPs, magainin-2 and melittin,
was investigated, showing that the concentrations required for membrane
permeabilization and the kinetics of the dye entrance differ significantly.
Our results are discussed in light of the proposed permeabilization
models of the two AMPs. The presented data demonstrate the potential
of this setup for the development of an on-chip screening platform
for AMPs
Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates
A number
of techniques has been developed and analyzed in recent
years to generate pore-spanning membranes (PSMs). While quite a number
of methods rely on nanoporous substrates, only a few use micrometer-sized
pores to be able to individually resolve suspending membranes by means
of fluorescence microscopy. To be able to produce PSMs on pores that
are micrometer in size, an orthogonal functionalization strategy resulting
in a hydrophilic surface is highly desirable. Here, we report on a
method to prepare PSMs based on the evaporation of a thin layer of
silicon monoxide on top of the porous substrate. PM-IRRAS experiments
demonstrate that the final surface is composed of SiO<sub><i>x</i></sub> with 1 < <i>x</i> < 2. The hydrophilic
surface turned out to be well suited to spread giant unilamellar vesicles
forming PSMs. As the method does not rely on a gold coating as frequently
used for orthogonal functionalization, fluorescence micrographs provide
information not only from the freestanding membrane areas but also
from the supported ones. The observation of the entire PSM area enabled
us to observe phase-separation in these membranes on the freestanding
and supported parts as well as protein binding and possible lipid
reorganization of the membranes induced by binding of the protein
Shiga toxin
Rheology of Membrane-Attached Minimal Actin Cortices
The
actin cortex is a thin cross-linked network attached to the
plasma membrane, which is responsible for the cell’s shape
during migration, division, and growth. In a reductionist approach,
we created a minimal actin cortex (MAC) attached to a lipid membrane
to correlate the filamentous actin architecture with its viscoelastic
properties. The system is composed of a supported 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine bilayer doped with the receptor
lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P<sub>2</sub>) to which a constitutively active mutant of ezrin, which is a direct
membrane–cytoskeleton linker, is bound. The formation of the
MAC on the supported lipid bilayer is analyzed as a function of increasing
PtdIns(4,5)P<sub>2</sub>/ezrin pinning points, revealing an increase
in the intersections between actin filaments, that is, the node density
of the MAC. Bead tracking microrheology on the membrane-attached actin
network provides information about its viscoelastic properties. The
results show that ezrin serves as a dynamic cross-linker for the actin
cortex attached to the lipid bilayer and that the stiffness of the
network is influenced by the pinning point density, relating the plateau
storage modulus <i>G</i><sub>0</sub> to the node density
of the MAC