Biomedical subjects

P Hinterdorfer

Publications and source records attributed to P Hinterdorfer.

15 recordsLinked to original sources

PubMed2002

Single molecule fluorescence and force microscopy.

The investigation of biomolecules has entered a new age since the development of methodologies capable of studies at the level of single molecules. In biology, most molecules show a complex dynamical behavior, with individual motions and transitions between different states occurring highly correlated in space and time within an arrangement of various elements. Recent advances in the development of new microscopy techniques with sensitivity at the single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are described here in terms of their applicability to the in vivo detection and visualization of molecular processes on surfaces, membranes, and cells. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultra-sensitive methodology as a general tool to study local processes and heterogeneities in living cells.

Cell Membrane↗

PubMed2001

Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1).

The skeletal muscle Ca2+ release channel (ryanodine receptor 1, RYR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type 1 from rabbit white muscle and adsorbed it to mica sheets with the cytoplasmic side facing up. Single receptors of uniformly distributed size and shape of 10-12 nm height and 40-50 nm width, and occasionally some aggregates were seen in contact mode AFM images. These immobilized RYR1 were specifically recognized by rabbit anti-RYR1 (antibody#8) with at least 30% efficiency, as measured by an enzyme immunoassay with goat-anti-rabbit. Single specific antibody-antigen recognition events were detected with AFM tips to which an antibody#8 was tethered. In linear scans, the occurrence of antibody-antigen binding showed significant lateral dependence, which allowed for the localization of binding sites with nm resolution. Variation of the loading rate in force spectroscopy experiments revealed a logarithmic dependence of the unbinding forces, ranging from 42 to 73 pN. From this dependence, a bond width of the binding pocket of L = 0.2 nm and a kinetic off-rate of koff = 12.7s(-1) was determined.

Animals↗

PubMed2001

Detection and characterization of single biomolecules at surfaces.

The investigation of bio-molecules has entered a new age since the development of methodologies capable of studies at the level of single molecules. In biology, most molecules show a complex dynamical behavior, with individual motions and transitions between different states, occurring as highly correlated in space and time within an arrangement of various elements. In order to resolve such dynamical changes in ensemble average techniques, one would have to synchronize all molecules, which is hard to achieve and might interfere with important system properties. Single molecule studies, in contrast, do not require pretreatment of the system and resume, therefore, much less invasive methodologies. Here, we review recent employments for the investigation of bio-molecules on surfaces, in which the high local and temporal resolution of two complementary techniques, atomic force microscopy and single molecule fluorescence microscopy, is used to address single molecules. Novel methodologies for the characterization of biologically relevant parameters, functions and dynamical aspects of individual molecules are described.

Biotechnology↗

PubMed2000

Cadherin interaction probed by atomic force microscopy.

Single molecule atomic force microscopy was used to characterize structure, binding strength (unbinding force), and binding kinetics of a classical cadherin, vascular endothelial (VE)-cadherin, secreted by transfected Chinese hamster ovary cells as cis-dimerized full-length external domain fused to Fc-portion of human IgG. In physiological buffer, the external domain of VE-cadherin dimers is a approximately 20-nm-long rod-shaped molecule that collapses and dissociates into monomers (V-shaped structures) in the absence of Ca(2+). Trans-interaction of dimers is a low-affinity reaction (K(D) = 10(-3)-10(-5) M, k(off) = 1.8 s(-1), k(on) = 10(3)-10(5) M(-1) x s(-1)) with relatively low unbinding force (35-55 pN at retrace velocities of 200-4,000 nm x s(-1)). Higher order unbinding forces, that increase with interaction time, indicate association of cadherins into complexes with cumulative binding strength. These observations favor a model by which the inherently weak unit binding strength and affinity of cadherin trans-interaction requires clustering and cytoskeletal immobilization for amplification. Binding is regulated by low-affinity Ca(2+) binding sites (K(D) = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca(2+) in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and communication.

Animals↗

PubMed2000

Optimal sensitivity for molecular recognition MAC-mode AFM

Molecular recognition force microscopy (MRFM) using the magnetic AC mode (MAC mode) atomic force microscope (AFM) was recently investigated to locate and probe recognition sites. A flexible crosslinker carrying a ligand is bound to the tip for the molecular recognition of receptors on the surface of a sample. In this report, the driving frequency is calculated which optimizes the sensitivity (S). The sensitivity of MRFM is defined as the relative change of the magnetically excited cantilever deflection amplitude arising from a crosslinker/antibody/antigen connection that is characterized by a very small force constant. The sensitivity is calculated in a damped oscillator model with a certain value of quality factor Q, which, together with load, defines the frequency response (unloaded oscillator shows resonance at Q > 0.707). If Q < 1, the greatest value of S corresponds to zero driving frequency omega (measured in units of eigenfrequency). Therefore, for Q < 1, MAC-mode has no advantage in comparison with DC-mode. Two additional extremes are found at omegaL = (1 - 1/Q)(1/2) and omegaR = (1 + 1/Q)(1/2), with corresponding sensitivities S(L) = Q2/(2Q - 1), S(R) = Q2/(2Q + 1). The L-extreme exists only for Q > 1, and then S(L) > S(R), i.e. the L-extreme is the main one. For Q > 1, S(L) > 1, and for Q > 2.41, S(R) > 1. These are the critical Q-values, above which selecting driving frequency equal to sigmaL or sigmaR brings advantage to MAC mode vs. DC mode. Satisfactory quality of the oscillator model is demonstrated by comparison of some results with those calculated within the classical description of cantilevers.

Journal Article↗

PubMed2000

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.

Journal Article↗

PubMed1999

Microscopy for recognition of individual biomolecules.

One frontier challenge in microscopy and analytical chemistry is the analysis of soft matter at the single molecule level with biological systems as most complex examples. Towards this goal we have developed two novel microscopy methods. Both employ highly specific molecular recognition schemes used by nature-the recognition of specific protein sites by antibodies and ligands. One method uses fluorescence labeled ligands for detecting single molecules in fluid systems like membranes (Fig. 1B). Unitary signals are reliably resolved even for millisecond illumination periods. The knowledge of the unitary signal from single molecules permits the determination of stoichiometries of component association (Fig. 3). Direct imaging of the diffusional path of single molecules became possible for the first time (Fig. 4). Using linear polarized excitation, the angular orientation of single molecules can be analyzed (single molecule linear dichroism, (Fig. 5), which opens a new perspective for detecting conformational changes of single biomolecules. In the other method, an antibody is flexibly linked to the tip of an atomic-force microscope. This permits the identification of receptors in multi-component systems. Molecular mapping of biosurfaces and the study of molecular dynamics in the ms to s range become possible with atomic force microscopy.

Antibodies↗

PubMed1999

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.

Antigen-Antibody Reactions↗

PubMed1998

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.

Aluminum Silicates↗

PubMed1997

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor.

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.

Calcium Channels↗

PubMed1997

A non-invasive method for the tight anchoring of cells for scanning force microscopy.

Use of scanning force microscopy (SFM) for high resolution imaging of cell surfaces requires the cells to be tightly attached to substrates. Imaging of loosely adhered RBL-2H3 cells enabled determination of the cell size and investigation of larger structures and pseudopodia but failed in resolving more detail. Immobilization under non-invasive conditions via flexible crosslinkers containing a hydrophobic anchoring group enhanced resolution enormously. The cells were tightly attached to the substrates and were not removed by shear forces up to 80 nN as determined in a flow through apparatus. Morphological structures and dynamic processes on cell surfaces were observed as well as structural changes after cell stimulation upon ionomycin treatment. Molecular or atomic resolution, however, was not attainable which is attributed to the displacement of the flexible cell surface due to shear forces arising from the scanning tip during contact mode.

Animals↗

PubMed1996

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Animals↗

PubMed1995

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes. At neutral pH, the orientation of the HA trimers in planar membranes is approximately perpendicular to the membrane. However, at the pH of fusion, the HA trimers are tilted 55-70 degrees from the membrane normal in the presence or absence of bound target membranes. In the absence of target membranes, the overall secondary structure of HA at the fusion pH is similar to that at neutral pH, but approximately 50-60 additional residues become alpha-helical upon the conformational change in the presence of bound target membranes. These results are discussed in terms of a structural model for the fusion intermediate of influenza HA.

Animals↗

PubMed1994

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.

Animals↗

PubMedDate not supplied

Single molecule microscopy of biomembranes (review).

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.

Animals↗