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Biomedical subjects

E Sackmann

Publications and source records attributed to E Sackmann.

At least 55 records · Page 3Linked to original sources
PubMed1996

Neural cell pattern formation on glass and oxidized silicon surfaces modified with poly(N-isopropylacrylamide).

Control over the adsorption of proteins and over the adsorption and spatial orientation of mammalian cells onto surfaces has been achieved by modification of glass and other silicon oxide substrates with poly(N-isopropylacrylamide) (PNIPAM). The functionalization of the substrates was achieved either by a polymer-analogous reaction of aminosilanes with reactive N-(isopropylacrylamide) (NIPAM)-copolymers and by copolymerization of NIPAM with surface-bound methacrylsilane. The obtained coatings were characterized by FT-1R, ellipsometry, and surface plasmon resonance measurements. The adsorption of two proteins-fibrinogen and ribonuclease A-on these surfaces was studied in situ by real time surface plasmon resonance measurements. The PNIPAM-grafted surfaces prepared by either chemical procedure inhibited the adsorption of both proteins. More importantly they prevented the adhesion of neuroblastomaXglioma hybrid cells cultured either in serum-free medium or in a medium containing serum proteins. Deep-UV irradiation was used to perform ablation processes and to create patterns permitting the examination of spatially controlled adhesion and growth of cells. This study showed that patterned ultrathin polymer films on glass are suitable substrates for controlling the interactions of cells with surfaces and are capable of directing the attachment and spreading of cells.

Acrylamides↗
PubMed1995

The actin-binding protein hisactophilin binds in vitro to partially charged membranes and mediates actin coupling to membranes.

The interaction of the actin-binding protein hisactophilin from Dictyostelium discoideum amoebae to partially charged lipid membranes composed of mixtures of L-alpha-dimyristoylphosphatidylcholine (DMPC) with L-alpha-dimyristoylphosphatidylglycerol (DMPG) and L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is studied by film balance experiments, microfluorescence, and lateral diffusion measurements at low ionic strengths (approximately 20 mM). Excess surface concentrations and adhesion energies of the protein are evaluated by the application of Gibbs law of surface excess as a function of charged lipid content. Protein expressed in E. coli lacking a myristic acid chain (EC-HIS) and natural protein with a fatty acid (DIC-HIS) isolated from Dictyostelium cells are compared. For mixtures of DMPG and DMPC, protein binding leads to an increase in lateral pressure of the monolayer (at constant area) and causes strong lipid immobilization pointing to partial penetration of the protein into the lipid layer. The natural protein causes a much stronger immobilization than does EC-HIS. For a given bulk concentration, the adsorbed protein/lipid molar ratio increases with the molar fraction chi PG of charged lipid but saturates at about 50 mol% of DMPG. Natural hisactophilin (DIC-HIS) binding to PIP2-containing monolayers is purely electrostatic at low bulk concentration cb, and protein penetration dominates only at cb > 68 nM. Fluorescence experiments demonstrate that the natural protein (DIC-HIS) can mediate the binding of monomeric actin or very small oligomers to membranes, showing that the adsorbed protein remains functional. In contrast, the recombinant hisactophilin (EC-HIS) can mediate only the membrane coupling of larger actin structures.

Actins↗
PubMed1995

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.

Amino Acid Sequence↗
PubMed1995

Measurement of erythrocyte membrane elasticity by flicker eigenmode decomposition.

We have studied the flickering of erythrocytes at wavelengths comparable to the cell dimension. To do this we have analyzed the edge fluctuations of the cell to a resolution of 5 nm by combining phase contrast microscopy with fast image processing. By measuring the edge excitations simultaneously at four orthogonal positions around the cell, the eigenmodes of equal azimuthal mode numbers m = 0,1,2 could be separated. From a continuous time sequence of 100 s of video frames taken at 40 ms time intervals, we determined the time-auto correlation function for the modes m = 0,1,2 and calculated their mean square amplitudes as well as their decay times tau m. To explain the results we also present the theoretically calculated energy eigenmodes of an erythrocyte, accounting for the constraint that the cell is in contact with the substrate along an annular ring, which agreed well with the experimental findings. We found that the softest mode is a "hindered translational" mode with m = 1 of the adhered cell, which is almost insensitive to the shear elastic modulus. Comparison of the calculated and measured amplitudes yielded an average value for the bending stiffness of kc = 4 x 10(-19) J, which is much larger than the value obtained by flicker analysis at short wavelengths (kc = 2.3 x 10(-20) J). It would, however, agree well with the value expected from the red cell membrane area compressibility modulus of K = 4.5 x 10(-1)N/m, which corresponds to a lipid bilayer containing approximately 50 mol % of cholesterol. In contradiction to our theoretical expectations we found that the flicker eigenmodes seemed not to be influenced by the membrane shear elasticity, which will be discussed in terms of an unusual coupling between the lipid bilayer and the cytoskeleton.

Biophysical Phenomena↗
PubMed1995

Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex.

Animals↗
PubMed1995

Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography.

Automated electron tomography is shown to be a suitable means to visualize the shape of phospholipid vesicles embedded in vitrified ice. With a slow-scan charge-coupled device camera as a recording device, the cumulative electron dose needed to record a data set of 60 projections at a magnification of 20,000X can be kept as low as 15 e-/A2 (or 1500 electrons/nm2). The membrane of the three-dimensionally reconstructed vesicles is clearly visible in two-dimensional sections through the three-dimensionally reconstructed volume. Some edges indicating a polygonal shape of the vesicles, frozen from the gel phase, are also clearly recognized. Because of the presently limited tilt angle range (+/- 60 degrees), the upper and lower "caps" of the vesicles (representing about 35% of the surface of the ellipsoidal particles) remain invisible in the three-dimensional reconstruction.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Hydration dependence of chain dynamics and local diffusion in L-alpha-dipalmitoylphosphtidylcholine multilayers studied by incoherent quasi-elastic neutron scattering.

Incoherent quasi-elastic neutron scattering is applied to study the local diffusion and chain dynamics of L-alpha-diplamiotylphosphatidylcholine molecules in oriented model membranes. Different motions are distinguished by changing the hydration of the multilayers as well as by measuring below and above the gel-to-liquid crystalline phase transition. The time range of the utilized time-of-flight spectrometer permits to observe two types of motion to be observed more closely: chain defect motions and the local diffusion of the whole molecule in its solvation cage. Oriented lipid membranes are a useful system for the observation of chain defects, as they can be macroscopically oriented, in contrast to most polymers. As a representative model for a chain defect a kink is chosen and the corresponding scattering functions are derived. The kink motion can explain the entire dynamics seen in the gel phase, and the lifetime of such a defect was found to be 10-15 ps, in good agreement with theoretical predictions. On the other hand the dynamics in the liquid crystalline phase cannot be explained even by a superposition of several kinks and thus requires the consideration of an additional motion: the local diffusion of the molecule in its solvation cage. The size of the solvation cage is increasing with multilayer hydration and reduced temperature. Particularly interesting in view of recent discussions about the origin of the short-range repulsive forces between membranes is the experimental finding of an out-of-plane motion with an amplitude of 1-1.5 A, which cannot be explained by the undulation of the whole membrane.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Self assembly of covalently anchored phospholipid supported membranes by use of DODA-Suc-NHS-lipids.

We present a novel preparation method for the self assembly of covalently anchored phospholipid supported membranes. The surface is gold covered by cysteamine. Vesicles containing DMPC and activated DODA-Suc-NHS-lipids assembled on this surface. The whole self-assembly process is monitored conveniently by Near Infrared Surface Plasmon Resonance (NIR-SPR). Comparing the data to those obtained by Ca(2+)-mediated vesicle fusion, confirmed this interpretation.

Dimyristoylphosphatidylcholine↗
PubMed1994

The mechanical properties of actin gels. Elastic modulus and filament motions.

To address large discrepancies reported in the literature, the viscoelastic properties of gels formed by purified actin filaments have been measured by five different techniques and five different instruments using actin preparations purified separately in four different laboratories. These measurements consistently showed that the elastic shear modulus of 2 mg/ml F-actin is on the order of several hundred pascals, and depends very strongly on the length of the filaments and on the history of the sample prior to measurement. Shortening of actin filaments with gelsolin and mechanical perturbations reduce the shear modulus to low values identical to some reported in the literature, indicating that such perturbations account for low shear moduli and poor responsiveness to filament modifying treatments reported previously. The structures of individual actin filaments within gels very similar or identical to those studied rheometrically were also examined by dynamic light scattering and fluorescence microscopy. Dynamic light scattering data were analyzed by a new method to confirm that actin filaments have no stable associations with each other and fluctuate in solution at a rate governed by the filament bending modulus or persistence length, determined to be approximately 10 microns. Fluorescence microscopy confirmed that applying even small shear stresses to F-actin can orient and rupture the filaments, and that in a minimally perturbed viscoelastic gel, long actin filaments are free to diffuse within a limit of constraints formed by their neighbors. These findings confirm that relatively isotropic F-actin networks are sufficiently strong to stabilize cells.

Actins↗
PubMed1994

Insertion of filamin into lipid membranes examined by calorimetry, the film balance technique, and lipid photolabeling.

The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studied in reconstituted lipid monolayers and bilayers. Protein-lipid interactions were investigated by differential scanning calorimetry, the film balance technique, and hydrophobic photoradiolabeling. For calorimetric assays, multilamellar vesicles (MLVs) and large unilamellar vesicles produced by the extrusion technique (LUVETs) were used. With MLVs, filamin induced a pronounced drop in phase transition cooperativity. Mixed DMPC/DMPG LUVETs showed a linear decrease of the main phase transition enthalpy and a significant shift in temperature for the solidus and liquidus lines with increasing mole fractions of reconstituted filamin. The insertion of native filamin into uncharged and negatively charged lipid monolayers was measured in time/area diagrams with the film balance technique. Finally, we have newly synthesized a highly sensitive lipid analogue, [125]TID-PC/16, which selectively labels membrane-embedded hydrophobic domains of proteins, and which proved to label filamin, supporting evidence that this protein partially inserts into the hydrophobic domain of liposomes.

Calorimetry, Differential Scanning↗
PubMed1994

The seventh Datta Lecture. Membrane bending energy concept of vesicle- and cell-shapes and shape-transitions.

The main objective of this lecture is to discuss the role of lipid-bilayer elasticity (1) for the self-organization of lipid/protein-bilayers (2) for the stabilization of domain structures and shapes of cell membranes and (3) for the control of shape transitions (e.g. bud- and pit-formation) and shape instabilities (vesicle fission). It is demonstrated that many complex shape transitions of cell membranes can be mimicked by single lipid bilayer vesicles by simply varying the area-to-volume ratio or by chemically induced bending moments suggesting that these processes are governed by the universal minimum bending energy concept of closed shells composed of stratified membranes. The essential role of the coupling between curvature and phase separation in mixed membranes for the formation and stabilization of local pits and buds or the fission of budded vesicles is demonstrated. Finally, we discuss the consequences of the pronounced thermally excited bending undulations of the hyperelastic membranes for the membrane tension, the material exchange at membrane surfaces and the control of the adhesion of vesicles (or cells) on solid substrates.

Biophysical Phenomena↗
PubMed1994

Direct imaging of reptation for semiflexible actin filaments.

According to the reptation model of polymer diffusion, a polymer chain exhibits snake-like motion through the entangled mesh of surrounding molecules, in which the undulations of the chain are restricted to a tube-like region. The reptation model can account for many of the dynamic properties of entangled polymer solutions and melts, and has received support from observations of block copolymer diffusion across an interface; but reptative motion has not previously been imaged directly. Here we report such a direct observation of reptation, obtained by video microscopy of fluorescently labelled single, semiflexible filaments of actin in a solution of unlabelled actin filaments. From the restricted thermal undulations of these filaments we can measure the diameter of the confining tube, and we also observe the characteristic thermally excited sliding of the filament out of the end of the tube. We find that the chain self-diffusion coefficient decreases approximately linearly as the filament length increases, in agreement with the reptation model.

Actins↗
PubMed1994

Lipid mono- and bilayer supported on polymer films: composite polymer-lipid films on solid substrates.

We report the deposition of lipid monolayers and bilayers on polyacrylamide films deposited by radical chain reaction onto solid substrates in aqueous solutions. Polymer films of various degrees of monomer density and cross-linking are prepared. Lateral diffusion and fluorescent probe permeation measurements yield insight into the continuity of the lipid layers and show that monolayers exposed to air are much less sensitive towards polymer heterogeneities than bilayers below water, which is explained in terms of the wetting laws. The diffusion studies of lipid and lipopeptide probes yield absolute values of the frictional coefficients between the lipid layer and the polymer films and allow one to estimate the surface viscosity of the polymer film. The potential applications of supported membranes on soft thin polymer films for the preparation of biofunctionalized surfaces or biocompatible receptive surfaces for biosensors are discussed.

Acrylic Resins↗
PubMed1994

Local measurements of viscoelastic moduli of entangled actin networks using an oscillating magnetic bead micro-rheometer.

A magnetically driven bead micro-rheometer for local quantitative measurements of the viscoelastic moduli in soft macromolecular networks such as an entangled F-actin solution is described. The viscoelastic response of paramagnetic latex beads to external magnetic forces is analyzed by optical particle tracking and fast image processing. Several modes of operation are possible, including analysis of bead motion after pulse-like or oscillatory excitations, or after application of a constant force. The frequency dependencies of the storage modulus, G'(omega), and the loss modulus, G''(omega), were measured for frequencies from 10(-1) Hz to 5 Hz. For low actin concentrations (mesh sizes epsilon > 0.1 micron) we found that both G'(omega) and G''(omega) scale with omega 1/2. This scaling law and the absolute values of G' and G'' agree with conventional rheological measurements, demonstrating that the magnetic bead micro-rheometer allows quantitative measurements of the viscoelastic moduli. Local variations of the viscoelastic moduli (and thus of the network density and mesh size) can be probed in several ways: 1) by measurement of G' and G'' at different sites within the network; 2) by the simultaneous analysis of several embedded beads; and 3) by evaluation of the bead trajectories over macroscopic distances. The latter mode yields absolute values and local fluctuations of the apparent viscosity eta(x) of the network.

Actins↗
PubMed1993

Interactions of liposomes and hydrophobically-modified poly-(N-isopropylacrylamides): an attempt to model the cytoskeleton.

The interactions of small unilamellar vesicles (SUV) and water-soluble copolymers were studied by fluorescence spectroscopy, differential scanning calorimetry (DSC) and quasi-elastic light scattering (QELS). The anchoring onto liposomal bilayer membranes of copolymers of N-isopropylacrylamide, N-(2-(1-naphthyl)ethyl)-N-n-octadecylacrylamide and or N-[4-(1-pyrenyl)butyl]-N-n-octadecylacrylamide (0.5 mol% of the octadecylacrylamide comonomer) was monitored by non-radiative energy transfer between excited naphthalene and pyrene. The anchoring process occurred on zwitterionic lecithin liposomes and on negatively charged phosphatidic acid liposomes, whether the bilayer was in the crystalline or the liquid-crystalline phase. Insertion of the copolymer octadecyl groups within crystalline bilayers was attributed to the presence of packing defects. Aqueous solutions of poly-(N-isopropylacrylamide) and of its hydrophobically-modified copolymers exhibit a lower critical solution temperature (LCST). The coil to globule collapse of the polymer chains which is known to occur as the aqueous solution is heated through the LCST, also took place when the copolymers were anchored onto vesicular bilayers. The copolymers remained anchored during this collapse and the liposomes were not destroyed. The process was thermo-reversible. Detailed aspects of the reversibility of the phenomenon depended on the relative values of the phase transition temperatures of the liposomes and of the polymer LCST.

Acrylic Resins↗
PubMed1993

Intersecting polymers in lipid bilayers: cliques, static order parameters and lateral diffusion.

We have modelled a macrolipid polymer composed of lipid molecules (monomers) embedded in a lipid bilayer or monolayer and polymerized via their polar groups. Because of fluctuations perpendicular to the plane of the bilayer, the polar region occupied by the polymer chain possesses sufficient space so that the polymer might exhibit 'self-intersection' if its conformational state is projected onto the plane of the bilayer/monolayer. We represent the plane of the bilayer/monolayer by a triangular lattice. Each site can be occupied by a monomer or be empty (and thus occupied by one of the unpolymerizable lipids which make up the bilayer/monolayer). A macrolipid is represented by a sequence of N monomers connected by N-1 bonds. Bonds may be either short (connecting nearest neighbour monomers) or long (between second neighbour monomers), in accord with the average properties of the spacers between the polymerized lipids. We have carried out computer simulation of this system using the Carmesin-Kremer bond stretching algorithm. Although no two monomers can occupy the same site, bonds may cross each other. We analyzed the dependence of and approximately N2vc and + approximately N2 sigma c, where Nsc and Nmc are the number of bond-crossings in the same macrolipid ('self-crossing') or in two different macrolipids ('mutual-crossing'). For single macrolipids, we confirmed that vc = 3/4 and have found that sigma c approximately 0.52, which we consider supports that sigma c = 1/2. For the dense case with monomer concentration, c = 0.72, we found that vc = 1/2 and that sigma c approximately 0.52 supports that sigma c = 1/2. In the semi-dilute regime (c = 0.2) we found crossover behaviour, although sigma c = 1/2. The total number of bond crossings thus scale like N, independent of concentration. We studied the connectivity of the system by calculating the weight averaged cluster, or 'clique', size. Cliques are defined as being composed of all macrolipids which exhibit at least one crossing bond with one other member of the clique. We found that while the average clique contains about two macrolipids at low concentrations, the clique size approaches the maximum possible value at high concentrations if the macrolipids are sufficiently long. In the latter case a transition appears to occur as the macrolipid length increases. This transition occurs at length = 40 when c = 0.72. These observations should have experimental consequences for the viscoelastic properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Simulation↗