14 research outputs found
Modulating the Lateral Tension of Solvent-Free Pore-Spanning Membranes
The plasma membrane of animal cells
is attached to the cytoskeleton,
which significantly contributes to the lateral tension of the membrane.
Lateral membrane tension has been shown to be an important physical
regulator of cellular processes such as cell motility and morphology
as well as exo- and endocytosis. Here, we report on lipid bilayers
spanning highly ordered pore arrays, where we can control the lateral
membrane tension by chemically varying the surface functionalization
of the porous substrate. Surface functionalization was achieved by
a gold coating on top of the pore rims of the hexagonal array of pores
in silicon nitride substrates with pore radii of 600 nm followed by
subsequent incubation with various <i>n</i>-propanolic mixtures
of 6-mercapto-1-hexanol (6MH) and <i>O</i>-cholesteryl <i>N</i>-(8′-mercapto-3′,6′-dioxaoctyl)Âcarbamate
(CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-<i>sn</i>-glycero-3-phosphocholine were prepared by spreading giant
unilamellar vesicles on these functionalized porous silicon nitride
substrates. Different mixtures of 6MH and CPEO3 provided self-assembled
monolayers (SAMs) with different compositions as analyzed by contact
angle and PM-IRRAS measurements. Site specific force-indentation experiments
on the pore-spanning membranes attached to the different SAMs revealed
a clear dependence of the amount of CPEO3 in the monolayer on the
lateral membrane tension. While bilayers on pure 6MH monolayers show
an average lateral membrane tension of 1.4 mN m<sup>–1</sup>, a mixed monolayer of CPEO3 and 6MH obtained from a solution with
9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m<sup>–1</sup>. From contact angle and PM-IRRAS results, the mole fraction of CPEO3
in solution can be roughly translated into a CPEO3 surface concentration
of 40 mol %. Our results clearly demonstrate that the free energy
difference between the supported and freestanding part of the membrane
depends on the chemical composition of the SAM, which controls the
lateral membrane tension
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
3D-Membrane Stacks on Supported Membranes Composed of Diatom Lipids Induced by Long-Chain Polyamines
Long-chain polyamines
(LCPAs) are intimately involved in the biomineralization
process of diatoms taking place in silica deposition vesicles being
acidic compartments surrounded by a lipid bilayer. Here, we addressed
the question whether and how LCPAs interact with lipid membranes composed
of glyceroÂphospholipids and glyceroÂglycolipids mimicking
the membranes of diatoms and higher plants. Solid supported lipid
bilayers and monolayers containing the three major components that
are unique in diatoms and higher plants, i.e., monoÂgalactosylÂdiacylglycerol
(MGDG), digalactosylÂdiacylglycerol (DGDG), and sulfoquinovosylÂdiacylglycerol
(SQDG), were prepared by spreading small unilamellar vesicles. The
integrity of the membranes was investigated by fluorescence microscopy
and atomic force microscopy showing continuous flat bilayers and monolayers
with small protrusions on top of the membrane. The addition of a synthetic
polyamine composed of 13 amine groups separated by a propyl spacer
(C3N13) results in flat but three-dimensional membrane stacks within
minutes. The membrane stacks are connected with the underlying membrane
as verified by fluorescence recovery after photobleaching experiments.
Membrane stack formation was found to be independent of the lipid
composition; i.e., neither glyceroglycolipids nor negatively charged
lipids were required. However, the formation process was strongly
dependent on the chain length of the polyamine. Whereas short polyamines
such as the naturally occurring spermidine, spermine, and the synthetic
polyamines C3N4 and C3N5 do not induce stack formation, those containing
seven and more amine groups (C3N7, C3N13, and C3N18) do form membrane
stacks. The observed stack formation might have implications for the
stability and expansion of the silica deposition vesicle during valve
and girdle band formation in diatoms
High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes
Pore-spanning
membranes (PSMs) provide a highly attractive model
system for investigating fundamental processes in lipid bilayers.
We measure and compare lipid diffusion in the supported and suspended
regions of PSMs prepared on a microfabricated porous substrate. Although
some properties of the suspended regions in PSMs have been characterized
using fluorescence studies, it has not been possible to examine the
mobility of membrane components on the supported membrane parts. Here,
we resolve this issue by employing interferometric scattering microscopy
(iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine)
(DOPC) bilayers prepared on holey silicon nitride substrates that
were either (i) oxygen-plasma-treated or (ii) functionalized with
gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion
in regions suspended on pores with diameters of 5 μm is found
to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol,
similar diffusion coefficients are obtained for both the suspended
and the supported regions, whereas for oxygen-plasma-treated surfaces,
diffusion is almost 4 times slower in the supported parts of the membranes.
We attribute this reduced diffusion on the supported parts in the
case of oxygen-plasma-treated surfaces to larger membrane–substrate
interactions, which lead to a higher membrane tension in the freestanding
membrane parts. Furthermore, we find clear indications for a decrease
of the diffusion constant in the freestanding regions away from the
pore center. We provide a detailed characterization of the diffusion
behavior in these membrane systems and discuss future directions
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
A DNA-Inspired Synthetic Ion Channel Based on G–C Base Pairing
A dinucleoside containing guanosine
and cytidine at the end groups
has been prepared using a modular one-pot azide–alkyne cycloaddition.
Single channel analysis showed that this dinucleoside predominantly
forms large channels with 2.9 nS conductance for the transport of
potassium ions across a phospholipid bilayer. Transmission electron
microscopy, atomic force microscopy, and circular dichroism spectroscopy
studies reveal that this dinucleoside can spontaneously associate
through Watson–Crick canonical H-bonding and π–π
stacking to form stable supramolecular nanostructures. Most importantly,
the ion channel activity of this G–C dinucleoside can be inhibited
using the nucleobase cytosine
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
Bright field and fluorescence microscopic analysis of CX+ and CX− tumor sublines (A), Fabry fibroblasts (B), and Daudi Burkitt's lymphoma cells (C).
<p>Cells were stained either with a relevant isotype-matched control antibody (isotype) or with an Hsp70 (cmHsp70.1-FITC, green) or Gb3 (CD77 plus Cy3-conjugated secondary antibody, red) specific antibody. The co-localization of Hsp70 and Gb3 is visualized in yellow as a merge of red and green in the lowest panel. Scale bar marks 20 µm. In comparison to CX+ and Fabry cells, the density of Gb3 on the cell membrane of Daudi cells is markedly increased, as indicated by a high mean fluorescence intensity of the CD77 staining. A double stain of Hsp70-FITC and Gb3-Cy3 indicates co-localization of both markers on the cell surface (lower right graph). Similar findings were obtained in experiments using the Colo+/Colo− tumor sublines (data not shown). On the right hand panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001925#pone-0001925-g004" target="_blank">Figure 4C</a>, representative flow cytometric profiles of Daudi cells are shown; the upper graph represents the double staining pattern of the isotype-matched antibodies IgM-PE and IgG1-FITC; the graphs below show single staining of Daudi cells with Hsp70-FITC (second graph) and CD77-PE (third graph). The fourth graph represents the double-staining pattern of Daudi cells using Hsp70-FITC and CD77-PE antibodies.</p
Globotriaosylceramide content is significantly higher in Hsp70 membrane-positive tumor sublines compared to their negative counterparts.
<p>Comparative analysis of the cell surface density of glycosphingolipids such as Gb3 (globotriaosylceramide, CD77 Ab), LacCer (lactosylceramide, CDw17 Ab), DoCer (dodecasaccharideceramide, CD65s Ab, data not shown), GalCer (galactosylceramide, MAB 342 Ab), Cer (ceramide, MID15B4 Ab), and GM1 (Cholera Toxin subunit B) in CX+/CX− and Colo+/Colo− tumor sublines. *Indicates values for Hsp70 membrane-positive (CX+, Colo+) and the corresponding Hsp70 membrane-negative (CX−, Colo−) tumor subline that are significantly different (P<0.03).</p
Binding of Hsp70-FITC, and BSA-FITC to viable Gb3 membrane-negative K562 and Gb3 membrane-positive Daudi cells.
<p>(A) Significant binding of Hsp70-FITC was observed to Daudi cells at a concentration of 12 µg/ml and 24 µg/ml. (B) BSA-FITC did neither bind to K562 nor to Daudi cells, at any of the tested concentrations. (C) Specific blocking of Hsp70-FITC binding by anti-Gb3 monoclonal antibody CD77. Daudi cells were kept either untreated or incubated with anti-Gb3 (CD77), or anti-ceramide (MID15B4) antibodies (5 µg/ml) for 30 min on ice prior to incubation with Hsp70-FITC (60 µg/ml, 30 min on ice) and analysis by flow cytometry.</p