Mechanical response of adherent giant liposomes to indentation with a conical AFM-tip.

Indentation of giant liposomes with a conical indenter is described by means of a tension-based membrane model. We found that nonlinear membrane theory neglecting the impact of bending sufficiently describes the mechanical response of liposomes to indentation as measured by atomic force microscopy. Giant vesicles are gently adsorbed on glassy surfaces via avidin-biotin linkages and indented centrally using an atomic force microscope equipped with conventional sharp tips mounted on top of an inverted microscope. Force indentation curves display a nonlinear response that allows to extract pre-stress of the bilayer T0 and the area compressibility modulus KA by computing the contour of the vesicle at a given force. The values for KA of fluid membranes correspond well to what is known from micropipet suction experiments and inferred from membrane undulation monitoring. Assembly of actin shells inside the liposome considerably stiffens the vesicles resulting in significantly larger area compressibility modules. The analysis can be easily extended to different indenter geometries with rotational symmetry.

Mechanical properties of giant liposomes compressed between two parallel plates: impact of artificial actin shells.

The mechanical response of giant liposomes to compression between two parallel plates is investigated in the context of an artificial actin cortex adjacent to the inner leaflet of the bilayer. We found that nonlinear membrane theory neglecting the impact of bending sufficiently describes the mechanical response of liposomes consisting of fluid lipids to compression whereas the formation of an actin cortex or the use of gel-phase lipids generally leads to substantial stiffening of the shell. Giant vesicles are gently adsorbed on glassy surfaces and are compressed with tipless cantilevers using an atomic force microscope. Force-compression curves display a nonlinear response that allows us to determine the membrane tension σ0 and the area compressibility modulus K(A) by computing the contour of the vesicle as a function of the compression depth. The values for KA of fluid membranes correspond well to what is known from micropipet-suction experiments and computed from monitoring membrane undulations. The presence of a thick actin shell adjacent to the inner leaflet of the liposome membrane stiffens the system considerably, as mirrored in a significantly higher apparent area compressibility modulus.

Collective behavior of Dictyostelium discoideum monitored by impedance analysis.

Dictyostelium discoideum cells respond to periodic signals of extracellular cAMP by collective changes of cell-cell and cell-substrate contacts. This was confirmed by dielectric analysis employing electric cell-substrate impedance sensing (ECIS) and impedance measurements involving cell-filled micro channels in conjunction with optical microscopy providing a comprehensive picture of chemotaxis under conditions of starvation.

Chemotaxis of Dictyostelium discoideum: collective oscillation of cellular contacts.

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

Lipopeptides derived from HIV and SIV mimicking the prehairpin intermediate of gp41 on solid supported lipid bilayers.

We present a universal mimetic approach of the prehairpin intermediate of gp41, which represents the active drug target for fusion inhibitors of HIV (human immunodeficiency virus) and SIV (simian immunodeficiency virus) based on membrane anchored lipopeptides. For this purpose, we have in situ coupled terminal cysteine-modified peptides originating from the NHR of SIV and HIV to a maleimide-functionalized DOPC bilayer and monitored the interactions with potential antagonists of the trimer-of-hairpin conformation C34 and T20 peptides by means of atomic force microscopy and ellipsometry. FT-IR analysis in conjugation with CD-spectroscopy of hydrated N36-lipopeptides, incorporated in multilamellar bilayer stacks was employed to investigate peptide conformation prior to antagonist binding. In contrast to solution studies substantial secondary structure formation of S-N36 after in situ coupling to the bilayer was found. We could show that S-N36-lipopeptide-aggregates in bilayers were selectively able to bind T20 or the corresponding C-peptides (C34) and similar results could be achieved by using H-N36 lipopeptides. It was found that T20 binding to coiled coil S-N36 lipopeptide assemblies was fully reversible at elevated temperatures, while T20 binds irreversibly to H-N36 bundles.