Hansgeorg Schindler

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of ∼50 nm, using ‘single dye tracing’. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid‐specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid‐ordered phase: the probe showed confined but fast diffusion, with high partitioning (∼100‐fold) and long residence time (∼13 s). The analogous probe with mono‐unsaturated chains diffused predominantly unconfined within the membrane. With ∼15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 μm (0.2–2 μm), covering ∼13% of total membrane area. Both the liquid‐ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent‐resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.

Single-molecule microscopy on model membranes reveals anomalous diffusion

The lateral mobility of lipids in phospholipid membranes has attracted numerous experimental and theoretical studies, inspired by the model of Singer and Nicholson (1972. Science, 175:720-731) and the theoretical description by Saffman and Delbrück (1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113). Fluorescence recovery after photobleaching (FRAP) is used as the standard experimental technique for the study of lateral mobility, yielding an ensemble-averaged diffusion constant. Single-particle tracking (SPT) and the recently developed single-molecule imaging techniques now give access to data on individual displacements of molecules, which can be used for characterization of the mobility in a membrane. Here we present a new type of analysis for tracking data by making use of the probability distribution of square displacements. The potential of this new type of analysis is shown for single-molecule imaging, which was employed to follow the motion of individual fluorescence-labeled lipids in two systems: a fluid-supported phospholipid membrane and a solid polymerstabilized phospholipid monolayer. In the fluid membrane, a high-mobility component characterized by a diffusion constant of 4.4 microns2/s and a low-mobility component characterized by a diffusion constant of 0.07 micron2/s were identified. It is proposed that the latter characterizes the so-called immobile fraction often found in FRAP experiments. In the polymer-stabilized system, diffusion restricted to corrals of 140 nm was directly visualized. Both examples show the potentials of such detailed analysis in combination with single-molecule techniques: with minimal interference with the native structure, inhomogeneities of membrane mobility can be resolved with a spatial resolution of 100 nm, well below the diffraction limit.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand‐specific receptor clustering in rafts

The glycosylphosphatidylinositol‐anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co‐receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcγ‐receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co‐clustering with Toll‐like receptor 4, Fcγ‐RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand‐specific cellular responses initiated by CD14.

Experimental observation of the negatively charged water dimer and other small (H2O)−n clusters

Beams of (H2O)−n and (D2O)−n have been produced by injecting low energy electrons into a supersonic expansion of water and heavy water seeded in rare gases. Clusters with n≥2, with the exception of n=4, have been observed. The size distribution can be separated into three groups (n=2, n=6–7, and n≥10), which may be associated with different types of electron binding. The n<10 result gives a new lower limit for the number of water molecules necessary to bind an electron; the n≥10 correspond to the n=11 threshold observed earlier in pure H2O expansions.

Antibody recognition imaging by force microscopy

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody–antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.

SIMULTANEOUS HEIGHT AND ADHESION IMAGING OF ANTIBODY-ANTIGEN INTERACTIONS BY ATOMIC FORCE MICROSCOPY

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Static and Dynamical Properties of Single Poly(Ethylene Glycol) Molecules Investigated by Force Spectroscopy

Molecular recognition force spectroscopy was employed to probe the mechanical and dynamical features poly(ethylene glycol) (PEG). His6 was covalently coupled to AFM tips via PEG for the specific recognition of NTA on the surface. Force-extension profiles of single molecules obtained in force-distance cycles were fitted with an extended Worm Like Chain (WLC) model with a quality of the fit σdata-fit/σdata of 1.3. The fit revealed a persistence length LP of 3.8 ± 0.02 A and an enthalpic correction term K0 of 1561 ± 33 pN. Amplitude-distance cycles were recorded with dynamical force microscopy. Fitting with the damped linear oscillator model, using values for the persistence length and the nonlinear spring constant from force-distance cycles, yielded a fit quality σdata-fit/σdata of 1.5. Force-distance cycles calculated from amplitude-distance cycles by integration nicely agreed with simultaneously measured force-distance cycles, and even yielded an improved signal to noise ratio. This shows that no dissipative and irreversible processes occur and that the force extension profile of PEG is determined by purely elastic behavior.

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

We have investigated whether a raft heterogeneity exists in human monocyte‐derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X‐100 (Triton) or Lubrol WX (Lubrol) detergent‐resistant membranes from cholesterol‐loaded monocytes were associated with the following findings: (i) Lubrol‐DRM contained most of the cellular cholesterol and at least 75% of Triton‐detergent‐resistant membranes. (ii) ‘Lubrol rafts’, defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline‐phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton‐ and Lubrol‐detergent‐resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI‐mediated lipid efflux and CDC42 were partially localized in Lubrol‐ but not in Triton‐detergent‐resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline‐phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol‐ as well as in Triton‐detergent‐resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1‐dependent and HDL3‐mediated lipid efflux.

Formation of planar bilayers from artificial or native membrane vesicles

The formation of planar membranes from vesicles has been used to study the acetylcholine receptor from Torpedo electric organ in planar bilayers formed from native membrane vesicles [l] or from reconstituted vesicles [2] and it allowed reconstitution of porin (or matrix protein) channels as they occur in ~sc~e~c~~~ coli outer membranes [3]. The objective of this report is to detail the technique, to identify underlying mechanisms, and to explain the prevailing requirement for successful membrane formation that vesicle radii should exceed 50 nm.

Data analysis of interaction forces measured with the atomic force microscope

The force-distance cycle mode of the atomic force microscope (AFM) allows for detection of interaction forces between the AFM-tip and a substrate (probe). This can either be a direct tip-sample interaction or an interaction between molecules coupled to the tip and probe, respectively. The interaction forces are typically in the range of a few pN to some hundred pN. In this article we describe algorithms for the analysis of force-distance cycles, to quantify interaction forces between tip and probe. Both, the direct tip-probe interaction as well as the interaction between specifically bound molecules are analyzed. The molecules bound to tip and probe have to be either long and flexible or have to be bound via a flexible cross linker. The algorithms are exemplified on direct tip-probe interactions and on unbinding events of cadherins which are bound via PEG-spacers to the AFM-tip and to the probe.