Elastische Eigenschaften von porenüberspannenden Lipidmembranen

Biologische Membranen sind kleine Fabriken, an denen unzählige Stoffe hergestellt werden, die für die Zelle lebenswichtig sind. Wie diese Stoffe erzeugt werden, wird schon lange von Wissenschaftlern diskutiert. Eine Vermutung besagt, dass sich spezielle Lipide zu kleinen Einheiten in der Membran zusammenfinden, um gemeinsam andere Substanzen herzustellen. Da die Membran flüssig ist, kann man sich diese Einheiten wie kleine Flöße vorstellen, die auf ihr schwimmen ("Rafthypothese").

Wie untersucht ein Wissenschaftler solche Prozesse? Unglücklicherweise sind die beteiligten Moleküle nur einige Nanometer groß, so dass selbst zehntausend von Ihnen aneinandergereiht gerade einmal den Durchmesser eines Haares erreichen. Mit bloßem Auge sind sie daher nicht zu erkennen. Auch ein normales Mikroskop hilft nicht weiter.

Mit Hilfe des sogenannten Kraftmikroskops ist es möglich zur Klärung dieser offenen Fragen beizutragen. Ein Kraftmikroskop besteht aus einer kleinen Spitze, die die zu untersuchende Oberfläche wie ein Computerscanner abführt und misst, welche Kräfte zwischen ihr und der Spitze auftreten. Da die Spitze an ihrem unteren Ende nur ein Atom dick ist, hat man mit dem Instrument die Möglichkeit auch sehr kleine Ausschnitte der Oberfläche zu untersuchen.

Im Experiment wurde eine Membran über ein winziges Loch gespannt und mit der Kraftmikroskopspitze verformt. Durch Schlagen dieser ''Nano-Trommel'' konnte untersucht werden, wie die Membran sich unter Verbiegung verhält. Ihr Verhalten lässt Rückschlüsse auf ihre Zusammensetzung zu, also auch wie sich die Lipide in der Membran anordnen. Vielleicht ist es damit einmal möglich, Vermutungen wie die Rafthypothese zu bestätigen oder zu widerlegen.

Veröffentlichungen zu diesem Thema (in Englisch)

Theorie

• How to determine local elastic properties of lipid bilayer membranes from atomic-force-microscope measurements: A theoretical analysis
  Davood Norouzi, Martin Michael Müller, Markus Deserno

  Measurements with an atomic force microscope (AFM) offer a direct way to probe elastic properties of lipid bilayer membranes locally: provided the underlying stress-strain relation is known, material parameters such as surface tension or bending rigidity may be deduced. In a recent experiment a pore-spanning membrane was poked with an AFM tip, yielding a linear behavior of the force-indentation curves. A theoretical model for this case is presented here which describes these curves in the framework of Helfrich theory. The linear behavior of the measurements is reproduced if one neglects the influence of adhesion between tip and membrane. Including it via an adhesion balance changes the situation significantly: force-distance curves cease to be linear, hysteresis and nonzero detachment forces can show up. The characteristics of this rich scenario are discussed in detail in this article.
  
  Phys. Rev. E, 74(6): 061914, 2006. Siehe auch cond-mat/0602662.
  Ausgewählt für das Virtual Journal of Biological Physics Research.

   

Experimente

• Local Membrane Mechanics of Pore-Spanning Bilayers
  Ingo Mey, Milena Stephan, Eva K. Schmitt, Martin Michael Müller, Martine Ben Amar, Claudia Steinem, Andreas Janshoff

  The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was studied by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nanoblack lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominates over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented.
  
  J. Am. Chem. Soc., 131(20): pp. 7031-7039, 2009.

   

• Elasticity Mapping of Pore-Suspending Native Cell Membranes
  Bärbel Lorenz, Ingo Mey, Siegfried Steltenkamp, Tamir Fine, Christina Rommel, Martin Michael Müller, Alexander Maiwald, Joachim Wegener, Claudia Steinem, Andreas Janshoff

  The mechanics of cellular membranes is governed by a non-equilibrium composite framework consisting of the semiflexible filamentous cytoskeleton and extracellular matrix proteins linked to the lipid bilayer. While elasticity information of plasma membranes has mainly been obtained from whole cell analysis, techniques that allow to address local mechanical properties of cell membranes are desirable to learn how their lipid and protein composition is reflected in the elastic behavior on local length scales. Here, we introduce an approach based on basolateral membranes of polar epithelial Madin-Darby canine kidney (MDCK) II cells, prepared on a highly ordered porous substrate that allows elastic mapping on a submicrometer length scale. A strong correlation between the density of actin filaments and the measured membrane elasticity is found. Spatially resolved indentation experiments carried out with atomic force and fluorescence microscope permit to relate the supramolecular structure to the elasticity of cellular membranes. It is shown that the elastic response of the pore-spanning cell membranes is governed by the local bending modules rather than the lateral tension.
  
  Small, 5(7): pp. 832-838, 2009.

   

• Mechanical Properties of Pore-Spanning Lipid Bilayers Probed by Atomic Force Microscopy
  Siegfried Steltenkamp, Martin Michael Müller, Markus Deserno, Christian Hennesthal, Claudia Steinem, Andreas Janshoff

  We measure the elastic response of a free-standing lipid membrane to a local indentation by using an atomic force microscope. Starting point is a planar gold-coated alumina substrate with a chemisorbed 3-mercaptopropionic acid monolayer displaying circular pores of very well defined and tunable size, over which bilayers composed of N,N,- dimethyl- N,N,- dioctadecylammonium bromide or 1,2 - dioleoyl - 3 - trimethylammonium - propane chloride were spread. Centrally indenting these 'nanodrums' with an atomic force microscope tip yields force-indentation curves, which we quantitatively analyze by solving the corresponding shape equations of continuum curvature elasticity. Since the measured response depends in a known way on the system geometry (pore size, tip radius) and on material parameters (bending modulus, lateral tension), this opens the possibility to monitor local elastic properties of lipid membranes in a well-controlled setting.
  
  Biophys. J., 91(1): pp. 217-226, 2006.