Kartierung der elastischen Eigenschaften von freitragenden artifiziellen und biologischen Membranen auf porösen Substraten
Final Report Abstract
Membrane mechanics is essential for many biological processes such as cell migration, adhesion, cell division, exo- and endocytosis as well as cell shape/volume regulations. The ability of a lipid bilayer to deform or dilate strongly depends on its chemical composition and the existence of reservoirs to accommodate area changes. Quantitative data on the elastic properties of membranes were obtained from non-local methods such as micropipette aspiration of giant liposomes or flicker spectroscopy. However, due to the mosaic nature of biological membranes, it is highly desirable to measure local elastic properties of lipid bilayers on sub-micrometer length scales. As yet, the lack of suitable model systems prevented the use of scanning force techniques to map the elastic properties of membranes in a defined manner. Conventional solid supported lipid bilayers can only be compressed but bending or stretching requires a second aqueous compartment. With the advent of pore-spanning lipid bilayers, new stable membrane models became available that allow to address nano– to micrometer sized free-standing membrane patches organized in a well-defined array. The primary goal of this DFG-funded project was to establish a procedure to access the local elastic properties of pore suspending membranes using force microscopy. The main emphasis lied on the high resolution mapping of artificial lipid bilayers as well as cellular membranes derived from epithelial cells on porous substrates. Furthermore, we were interested in measuring the impact of external physical and biochemical stimuli on the mechanical properties of free–standing membranes. We established a variety of preparation protocols that allowed us to form artificial pore spanning bilayers on highly ordered porous silicon substrates with pore radii ranging from 225-600 nm. Additionally, we also used substrates with smaller pore size based on anodized alumina substrates. Three types of bilayers were distinguished: i) membranes originating from rupturing of GUVs on hydrophilic substrates rendering the bilayers essentially solvent–free and symmetric, ii) rupturing of GUVs on hydrophobic self-assembled monolayers forming hybrid bilayers on the pore rims, and iii) painted nano–BLMs on hydrophobic self-assembly monolayers. Axisymmetric force indentation experiments reveal that on larger pore sizes the restoring force originates mainly from pre–stress and lateral tension, while stretching and bending plays a minor role. Generally, we found that hybrid bilayers produce a larger pre–stress compared to solvent-free symmetric bilayers. Local bending modules could be mapped by following a combined strategy based on nanotube pulling in combination with indentation experiments permitting to determine lateral and bending module at the same spot quasi–simultaneously within a single force cycle. Alternatively, but less tractable is the use of ultra–small pores that produce very large curvature changes upon indentation. External stimuli such as addition of short chain alcohols reduce both the pre–stress of the bilayers as well as the bending modules considerably. Pore spanning apical as well as basal membranes derived from confluent epithelial monolayers (MDCK II) were successfully prepared and subjected to force indentation experiments allowing to access site specific bending modules and lateral tension. We found a strong correlation between lateral tension and the presence of actin filaments attached to the membrane fragments using overlays of AFM topography, fluorescence microscopy, and images displaying the local lateral tension of each individual pore.
Publications
- (2009). Elasticity mapping of apical cell membranes. Soft Matter 5, 3262–3265
Fine, T.; Mey, I.; Rommel, C.; Wegener, J.; Steinem, C.; Janshoff, A.
- (2009). Elasticity mapping of pore suspending native cell membranes. Small 5, 832–838
Lorenz, B.; Mey, I.; Steltenkamp, S.; Fine, T.; Rommel, C.; Mueller, M.M.; Maiwald, A.; Wegener, J.; Steinem, C. Janshoff, A.
- (2009). Local membrane mechanics of pore-spanning bilayers. J. Am. Chem. Soc. 131, 7031–7039
Mey, I.; Stephan, M. Schmitt, E.K.; Mueller, M.M.; Ben-Amar, M.; Steinem, C.; Janshoff, A.
- (2010). Cell adhesion on ordered pores: consequences for cellular elasticity. J. Adh. Sci. Technol. 24, 13-14, 2287–2300
Janshoff, A.; Lorenz, B.; Pietuch, A.; Fine, T.; Tarantola, M.; Steinem, C.; Wegener, J.
- (2010). Viscoelasticity of pore-spanning polymer membranes derived from giant polymerosomes. Soft Matter 6, 2508–2516
Kocun, M.; Mueller, W.; Maskos, M.; Mey, I.; Geil, B.; Steinem, C.; Janshoff, A.
- (2011). Orthogonal functionalization of nanoporous substrates: control of 3D surface functionality. ACS Appl. Mater. Interfaces 3, 1068–1076
Lazzara, T.; Kliesch, T.; Janshoff, A.; Steinem, C.
- (2011). Preparation of solvent-free, pore-spanning lipid bilayers: modeling the low tension of plasma membranes. Langmuir 27, 7672–7680
Kocun, M.; Lazzara, T.D.; Steinem, C.; Janshoff, A.
- (2011). Separating Attoliter-Sized Compartments using Fluid Pore-Spanning Lipid Bilayers. ACS Nano 5, 6935–6944
Lazzara, T.D.; Carnarius, C; Kocun, M.; Janshoff, A. Steinem, C.
