New insights into galvanotaxis and other directed cell movements: an analysis of the translocation distribution function.
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Animals↗
Biomedical subjects
H Gruler
Publications and source records attributed to H Gruler.
Interaction between enteroviruses and human endothelial cells in vitro. Alterations in the physical properties of endothelial cell plasma membrane and adhesion of human granulocytes.
Fluorescent molecular probes were used for study of the interaction between enteroviruses (Echo 9, Echo 12, and Coxsackie B3 virus) and human endothelial cells in monolayer culture. With the use of the monomer-excimer method with pyrene decanoic acid it was shown that a marked dose-dependent restructuring of the plasma membrane occurred following addition of virus to the endothelial cells. This took the form of an increase in the lipid surface available to the lipophil reporter molecules, probably due to an alteration in the domain structure of the plasma membrane caused by insertion of virus capsid proteins. Experiments with diphenylhexatriene indicated that the enteroviruses had only a slight tendency to make the plasma membrane of the endothelial cell more fluid. Concomitant with these alterations in the biophysical properties of the membrane, a virus-induced increase in granulocyte adherence to the endothelial cells was observed for all three enteroviruses studied. Possible mechanisms for this elevated adherence are discussed, as well as the significance of the results for the phenomenon of virus-induced granulocytopenia.
Cell Adhesion↗
Cell movement analysis in a necrotactic assay.
The methods of statistical physics have been applied to analysis of cell movement. Human leukocytes (granulocytes) were observed using time-lapse photography. The paths of the migrating cells were determined. The chemokinetic response at 35 degrees C is described by the diffusion constant (D = 233 micron2/min) and the track velocity (25 micron/min). A time-dependent chemotactic gradient is created by killing an erythrocyte by an intense laser flash. The chemotactic response at 35 degrees C is described by the degree of polar orientation (P1 = 0.85), the track velocity 24 micron/min, and the drift velocity towards the necrotactic source (v parallel = 20 micron/min). The track velocity as well the drift velocity show a broad distribution. The half-width of the velocity distribution. The half-width of the velocity distribution is approximately 5 micron/min. Cell movement can be described by elementary moving states. The characteristic time of the internal clock of the migrating cell is approximately 0.5 min. We found that the information transfer from the necrotactic gradient to the migrating cell is 1 bit per change in directed movement. A migrating cell cannot be stimulated within a period of approximately 10 s after the last decision to adapt a new moving direction.
Biophysical Phenomena↗
Physical bases for defining terms used to describe cell movement phenomena.
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Biophysical Phenomena↗
Analysis of cell movement.
The methods of statistical physics have been applied to the analysis of cell movement. Human leukocytes (granulocytes) were observed using time-lapse photography. The center of gravity of a cell, variations of cell shape, and cell orientation were investigated. This analytical description leads to a better understanding of cell movement. Stationary motion of a cell is described by the anisotropy of the cell shape. The cell displacement can be characterized by three different types of movement: The persistent mode where the cell moves away from an arbitrary chosen origin with its track velocity. The diffusion mode where the cells become dispersed in space by a random walk process. The drift mode where the cell moves with a drift velocity, v parallel, in a concentration gradient of chemoattractant molecules. The chemokinetic response is described by the diffusion constant D (= 240 microns2/min) and the track velocity vc (= 30 microns/min). The chemotactic response is described by the degree of orientation P1 (= 0.8), which is identical with the McCutcheon index and the chemotropism index. Cell movement can be described by elementary moving states, and the life time of such a moving state is 0.5 min. The survival probability of the moving state is determined by an internal program. It is not described by a stochastic process. The angular change in moving direction is also programmed, as the square root of the mean square angular change is +/- 50 degrees. The plus and minus direction are equally probable in a chemokinetic response. However, in a chemotactic assay the plus and minus directions are not equally probably. We found that the information transfer from the chemotactic gradient to the migrating cell is 1 bit per change in moving direction. A disturbance in this information transfer leads to an order-disorder transition. Furthermore, we found that the migrating cell exhibits a directional memory of 75 s.
Biophysical Phenomena↗
Echo 9 virus-induced order-disorder transition of chemotactic response of human polymorphonuclear leucocytes: phenomenology and molecular biology.
By means of functional, morphological, and biophysical methods the in vitro interaction of Echo virus, type 9, strain A. Barty with human polymorphonuclear leucocytes (PMNs) was investigated and analyzed by statistical methods. Control cells and virus-treated PMNs (15 min, 37 degrees C; PMN: virus (pfu)-ratio ranging from 1:1 to 1:50) were exposed to a chemotactic gradient (N-formylmethionyl-leucylphenylalanine = f-Met-Leu-Phe, 10(-8) M/mm) in a Zigmond chamber. Whereas the track velocity of the moving PMNs was not affected by the virus, the degree of orientation of virus-treated PMNs declined in a way dependent on the viral dose and on the time of PMN:virus interaction, resulting in a shift from chemotactic to chemokinetic response. This virus-induced order-disorder transition of chemotactic response can be described by a logarithmic law in analogy to the Weber-Fechner law. Parallel to the functional disturbances, virus-induced changes of cell shape, which could be confirmed by additional light and electron microscopy techniques, were also detected using statistical analysis of cytological data (median cell size, anisotropy of cell shape) by means of two-dimensional histograms. To investigate f-Met-Leu-Phe- or/and Echo 9 virus-induced PMN-cell membrane changes, the monomer-excimer technique with pyrenedecanoic acid as fluorescent probe was applied, which gives information about structural changes of the cell membrane. Addition of the chemotactic peptide (10(-8) M) to control PMNs resulted in a higher rate of excimer formation obviously due to the formation of new functional (receptor) units (= activated cell membrane). Echo 9 virus exhibited an opposite effect. Quantitative analysis of these results revealed that the f-Met-Leu-Phe-induced cell membrane changes were extinguished by the addition of 2 pfu Echo 9 virus. So far, we have additional indicators of a virus-induced order-disorder transition of chemotactic response of human PMNs on a molecular biological level.
Biophysical Phenomena↗
The analysis of nondirected cell locomotion.
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Cell Movement↗
F-Met-Leu-Phe and echo 9 virus interaction with human granulocytes. Changes of cell membrane structure.
Biophysical and biochemical methods were applied for investigation of cell membrane properties of human polymorphonuclear leukocytes (PMNs) exposed to the chemotactic peptide N-formylmethionyl-leucylphenylalanine (f-Met-Leu-Phe) and echovirus type 9, strain A, Barty. Steady-state fluorescence depolarization with diphenylhexatriene demonstrated no gross changes of the total membrane fluidity under the different experimental conditions. However, by means of the monomer-excimer technique with pyrenedecanoic acid (PDA), significant changes of the local membrane structure were detected for both agents. As demonstrated by a higher excimer ratio, the membrane area available for the PDA molecules was restricted by f-Met-Leu-Phe. This effect was dependent on the dose and on the time of interaction of the chemotactic peptide. These experimental findings were explained by the formation of functional receptor units ("activated membrane"). Echo 9 virus exhibited the opposite effect, characterized by a higher ratio of monomers, which also depended on the viral dose and the time of virus-PMN interaction. These virus-induced findings were explained by the dissolution of functional receptor units. Consecutive exposure of the PMNs to f-Met-Leu-Phe and echovirus, or vice versa, demonstrated a virus-predominant effect on the membrane structures.
Cell Membrane↗
Analysis of the directed and nondirected movement of human granulocytes: influence of temperature and ECHO 9 virus on N-formylmethionylleucylphenylalanine-induced chemokinesis and chemotaxis.
The directed movement of human polymorphonuclear leukocytes (PMN) in a plane (Zigmond chamber assay) is described by a statistical model. We demonstrate that (a) the movement of a single cell is a superposition of a directed and a random movement, and (b) the degree of orientation, P1, of moving cells in a chemotactic gradient can be determined either by the time average of a single cell or by the average of movement of multiple cells at a fixed time (Ergoden hypothesis). However, an homogeneous cell population is a necessary condition. P1, which is identical with the McCutcheon index, is derived from the measured angular distribution function of moving cells. The statistical model allows one to distinguish between chemotaxis and chemokinesis. Applying this model to the temperature-dependent changes of cell movement, we found that P1 = 0.82 (37 degrees C) decreased to P1 = 0.4 (22 degrees C). The average speed of moving cells exhibits a very strong temperature-dependent variation from 30 microns/min (37 degrees C) to 5 microns/min (22 degrees C), indicating a different temperature dependence of chemotaxis and chemokinesis. At a fixed temperature (37 degrees C) the stability of the chemotactic gradient can also be checked by the angular distribution function. In addition, this model was applied to investigate the enteric cytopathogenic human orphan, strain 9 (ECHO 9) virus-induced disturbances of cell movement. We found: (a) The average speed of cell movement is not affected by the virus. (b) The degree of orientation is not affected for virus doses below a critical virus dose, ao (virus/PMN = 0.8:1). (c) The degree of orientation above this critical value exhibits a time- and virus-dose-dependence. (d) At a fixed viral dose, the time-dependent decrease of P1 is described by an exponential law (virus/PMN = 5:1, the characteristic time is 110 min). (e) This characteristic time investigated as a function of viral dose results in a logarithmic law analogous with the Weber-Fechner law. These findings indicate that only chemotactic and not chemokinetic response is disturbed by ECHO 9 virus.
Cell Movement↗
[Changes in the membrane fluidity of human neutrophilic granulocytes under the effect of a chemoattractant (FMLP) and of echo virus, type 9, A. Barty strain].
Preincubation of human polymorphonuclear leucocytes (PMNs) with different concentrations of Echo virus, type 9, strain A. Barty results in viral dose- and time-dependent inhibition of chemotactic cellular response to chemoattractant (N-formylmethionylleucylphenylalanine = FMLP). In the present experiments, by means of biophysical methods using excimer forming lipids (pyrendecanoic acid) the influence of FMLP and Echo 9 virus on membrane fluidity of PMNs was investigated. It is shown that the increase of membrane fluidity elicited by FMLP is reduced by the virus in a dose dependent manner. These results indicate virus-induced disturbance of molecular architecture and failure of natural signal processing of PMN-membranes. This biophysical method, therefore, can be used as a functional histochemical method to demonstrate functional defects in membranes of living cells.
Chemotaxis, Leukocyte↗
Selective inhibition of human neutrophilic chemotaxis by echo virus, type 9. Virus-induced changes in membrane fluidity.
By means of the excimer-forming lipid technique changes of membrane fluidity of human PMNs exposed to a chemoattractant (FMLP) and/or Echo virus, type 9, strain A. Barty has been investigated. It was shown that preincubation of PMNs with Echo 9 virus results in a viral dose-dependent decrease of membrane fluidity of granulocytes exposed to FMLP. The virus-induced rigidity of cell membrane causes obviously a disturbance in the aggregation of membrane particles to form receptors, and thus can lead to a failure of regular processing of chemotactic signals.
Chemotactic Factors↗
Quantitative picture analysis of freeze-fracture electron-micrographs.
A method of three-dimensional reconstruction of the surface profile of artificial and natural membranes from freeze quenched electron micrographs is presented. The direct relation between the Pt-layer thickness and the local orientation of the membrane allows a reconstruction of the surface. The efficiency of this method is demonstrated on the quantitative analysis of some fine structures. These essential results are: 1. In the low resolution observation structural elements of a yeast cell were quantitatively described, (i) The diameter of a yeast cell is determined (4.2 microgram). (ii) The cell wall thickness is measured (150 nm). (iii) The dimension of cell wall incapsulated vesicles is determined (60-80 nm). (iv) The damlike protrusion in the plasma membrane has a triangular cross section. The height is 23 nm and the half width 50 nm. The particle assembly in the damlike protrusion is in a crystalline state. The change in surface curvature is probable due to a phase separation of a biaxial cluster in an uniaxial membrane. (v) Membrane bound particles can be distinguished by their surface profiles. 2. The resolution of surface profiles is limited by the size of the platinum grain. An average procedure can lead to a resolution of 0.2 nm. This increase is resolution can be understood with the uncertainty relation: The uncertainty of the profile in one dimension times the uncertainty in the other dimension (averaging length) is the area of the platinum grain. The monolayer thickness of dipalmitoyl phosphatidyl choline and dimyristoyl choline are distinguishable 2.6 +/- 0.2 nm and 2.4 +/- 0.2 nm respectively. The surface profile of a two-dimensional crystal in the membrane of a yeast cell can be determined with high accuracy. The two profiles of the inner and outer monolayer do not fit exactly together. A part of the membrane bound particle is pulled out of the monolayer during the fracturing procedure. 3. The third part investigates special fluctuation of the surface. (i) The mixture of dipalmitoyl phosphatidyl choline and dioleyl phosphatidyl choline shows a periodic structure. The fluctuation besides this periodicity can be explained by a spinodal decomposition during cryofixation. (ii) The fluctuation of a periodic structure can also be induced by thermal motion. The fluctuation of dimyristol phosphatidyl choline quenched from a temperature between the pre- and maintransition determines only one kind of elastic constant. This curvature elastic constant is in the order of 10(-20) Joule. (iii) The fluctuation of the particle density can be related with the particle-particle compressibility. We choose the clusters induced by polylysine in a membrane with charged and uncharged lipids as particles. The compressibility is in the order of 10(-6) Newton/m which is comparable to those of a monolayer in a gaseous state.
Cell Membrane↗
Three dimensional microscopic surface profiles of membranes reconstructed from freeze etching electrol micrographs.
A method of three-dimensional reconstruction of the surface profile of artificial and natural membranes from freeze quenched electron micrographs is presented. The method is based on the analysis of the variation in thickness of platinum layers, deposited under an oblique angle. In essence, it is reminiscent of the method of Eratosthenes to measure the earth's radius. The thickness of etch-like protrusions of membranes could be determined to an accuracy of about 3 A. True distances on curved surfaces rather than projections of distances are obtained. The method has been applied to both model membranes and biological membranes. The essential results are: 1. Detailed information on the symmetry and the molecular structure of the crystalline phases of dimyristoyl phosphatidylcholine was obtained. The microscopic surface profile of the ripple structure observed between the pretransition and the main transition was analysed. In accordance with a previous model we found that the ripple structure is caused by the spontaneous curvature of the monolayers. The surface profiles of the ripple structure and of the low temperature biaxial phase could be clearly distinguished. 2. The sizes and shapes of lipid domains formed by both thermically and charge-induced lateral phase separation were determined. This showed that the visual inspection of electron micrographs may lead to a considerable underestimation of the domain size. Conclusions may be drawn concerning the different phases formed upon lateral phase separation. 3. As a biological example, yeast cell membranes were studied. The method allows one to distinguish between different membrane-bound proteins by measuring the width-to-height ratio of the particles. The deformation of the lipid layer in the environment of the proteins may be determined. This deformation contains information about lipid-mediated long-range interactions between membrane proteins.
Freeze Etching↗
Chemoelastic effect of membranes.
The elastic theory of a uniaxial membrane in an asymmetric environment predicts a spontaneous splay deformation. This spontaneous curvature of the membrane is discussed by the intrinsic splay of the membrane molecules (e.g. wedge shaped molecules) and their polar orientation. The chemoelastic effect if the polar orientation induced by the asymmetric environment in connection with the intrinsic splay. This effect is also discussed for polyelectrolytes where a small change of pH (approximately 0.1) can lead to a spontaneous curvature of 10(4) cm-1. The actual shape of red blood cells can be explained by the spontaneous splay and a change in environment induces the change in shape of these cells. A model is proposed for two conical bodies swimming in a uniaxial membrane which interact with each other through elastic coupling. The force between the bodies can be either attractive or repulsive. As an example of this model clustering of proteins is discussed.
Elasticity↗
On domain structure and local curvature in lipid bilayers and biological membranes.
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Cholesterol↗
Chemokinesis and necrotaxis of human granulocytes: the important cellular organelles.
The directed and non-directed locomotion of human polymorphonuclear leukocytes on a glass surface was compared to Brownian and drift motion. The average track velocity was measured under different conditions. The track velocity of colchicine treated cells was the same as control cells. However, cytochalasin B treated cells and cytokineplasts had a reduced track velocity compared with the control cells. The non-directed locomotion was investigated by measuring the mean square displacement as a function of time. The diffusion constant, D, which quantitates the random walk process, and the characteristic time, tau, which governs the migration of the cell, was calculated. The value of the diffusion constant depended on the cell treatment: For control cells 261 micron2/min, for colchicine treated cells 145 micron2/min, for cytochalasin B treated cells 55 micron2/min, and for cytokineplasts 47 micron2/min. The characteristic time was about 40 s. The measurement showed that the nondirected locomotion can be described by the Brownian motion. The directed locomotion was investigated by a necrotactic assay and quantitated by the McCutcheon index. This index was for control cells 0.85 +/- 0.07, for colchicine treated cells 0.8 +/- 0.07, and for cytokineplasts 0.75 +/- 0.1. The measurement showed that the directed locomotion can be described by a process which is called drift mode. From this method of analysis it was determined that the important organelles of the cell for the directed and the non-directed locomotion are: (i) A part of the plasma membrane, (ii) the microfilaments, and (iii) an unstructurated part of the cytoplasme. The microtubules of the cell are only of minor importance for the directed and the non-directed locomotion.
Cell Movement↗
Cell movement and symmetry of the cellular environment.
The movement of micro-organisms was investigated in different cellular environments. The type of movement was described in terms of the symmetry of the cellular environment: (i) Random movement - isotropic symmetry of the environment, (ii) contact guidance - apolar symmetry of the environment and (iii) directed movement as chemotaxis and galvanotaxis - polar symmetry of the environment. To quantitate cell movement it was necessary to parameterize the environment as well as the cell movement by observables. The random movement was quantitated by the diffusion coefficient. The contact guidance of the nematic type and the contact guidance on a bent surface were quantified by an apolar order parameter. The contact guidance constant for fibroblast on a glass cylinder was 87.5 microns. The directed movement was quantified by a polar order parameter. Dose-response curves were derived and compared for different types of cells: Chemotaxis of granulocytes KCT-1 = 2.2 mm for 10 microM f-Met-Met-Met, galvanotaxis of granulocytes KG-1 = 0.2 V/mm, galvanotaxis of fibroblast KG-1 = 0.28 V/mm, galvanotaxis of spermatozoids of bracken fern KG-1 = 0.024 V/mm.
Cell Movement↗
Description of cell adhesion by the langmuir adsorption isotherm.
The adhesion of granulocytes to a glass surface is both theoretically and experimentally investigated. The basic results are: (i) The adherence process of cells to a surface can be described by the Langmuir adsorption isotherm which also holds for the adsorption of molecules to a substrate. (ii) The granulocytes do not interact with each other during the adsorption/desorption process. (iii) The equilibrium constant at 37 degrees C for granulocytes adsorption to the glass surface is 170 cells/mm3 = 1.7 x 10(5) cells/ml. (iv) The equilibrium constant increases with decreasing temperature. (v) The equilibrium constant is a function of the chemokinetic/chemotactic stimulus f-Met-Leu-Phe. (vi) The desorption rate is also a function of the chemokinetic/chemotactic stimulus (kde-1 = 71.5 min for 1 nM and 330 min for 1 microM f-Met-Leu-Phe). (vii) The adsorption rate is a function of the chemokinetic/chemotactic stimulus and the cellular bulk concentration (kad-1.cv-1 = 104 min for 1 nM and 8 min for 1 microM f-Met-Leu-Phe, and cV = 4000 cells/mm3 = 4 x 10(6) cells/ml). (viii) The total number of cells which can bind to the surface is independent of temperature (and of the chemokinetic/chemotactic stimulus). (ix) The high affinity receptor site is responsible for the adherence (Kf-Met- = 1.8 nM f-Met-Leu-Phe). (x) The chemokinetic/chemotactic stimulated adherence is a cooperative process on a molecular level. (xi) The cellular adsorption/desorption process is a rate controlled process. The thermodynamic description of the adsorption/desorption process failed.
Adsorption↗