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E L Elson

Publications and source records attributed to E L Elson.

At least 19 recordsLinked to original sources
PubMed1992

Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.

Actin Cytoskeleton↗
PubMed1992

Kinetics and mechanism of the folding of cytochrome c.

The reversible folding of cytochrome c in urea at pH 4.0 was investigated by repetitive pressure perturbation kinetics and by equilibrium spectroscopic methods. Two folding reactions were observed in the 1 ms to 10 s time range. The rates and amplitudes of these reactions depend on urea concentration in a complex manner, which is different for each process. The absorbance spectra of the kinetic amplitudes of the two reactions also differ from each other. A model with a three-state mechanism can quantitatively account for all of the kinetic and equilibrium data, and it enables us to determine the rate constants and volume changes of the two steps. If a rapid protonation step is added to the mechanism, the analysis can be extended to calculate the pH dependence of the rate and amplitude of the faster folding step. This pH dependence is in excellent agreement with previously published data [Tsong, T. Y. (1977) J. Biol. Chem. 252, 8778-8780]. Kinetic experiments in the 695-nm band show clearly that the axial ligand methionine-80 is involved in the slow folding process and the other axial ligand, histidine-18, is involved in the fast process. Additional experiments with a cyanogen bromide fragment of the protein, and fluorescence detection of the folding kinetics of the intact protein, support an interpretation of the model in terms of known structural elements of cytochrome c. This work provides new information about the mechanism of the folding of cytochrome c, resolves conflicts in earlier interpretations, and demonstrates the applicability of the repetitive pressure perturbation kinetics method to protein folding.

Animals↗
PubMed1992

Surface particle transport mechanism independent of myosin II in Dictyostelium.

Cellular locomotion could be driven by the rearward transport of membrane-bound particles observed on motile fibroblasts, keratinocytes and neuronal growth cones. A force propelling free surface particles backwards could move the cell forwards if the particles were anchored to a rigid substratum. During capping, myosin II ('double-headed' myosin) draws crosslinked membrane proteins to the rear of a cell. The mhcA- mutant of the amoebal stage of the slime mould Dictyostelium discoideum, in which the myosin II gene has been deleted, cannot cap surface particles but can crawl along the substratum. Thus, the mechanism driving capping is not essential for locomotion. We show here that the null mutant is capable of a different type of active rearward transport, independent of myosin II and distinct from capping. The transported particles on mhcA- cells follow parallel paths. In the wild-type Ax2 strain, myosin II causes particles to converge towards a focal point and significantly increases the velocity of transport behind the leading edge of the cell.

Actins↗
PubMed1992

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).

Actins↗
PubMed1991

Actin tracks.

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Actin Cytoskeleton↗
PubMed1991

Single particle tracking. Analysis of diffusion and flow in two-dimensional systems.

Analysis of the trajectories of small particles at high spatial and temporal resolution using video enhanced contrast microscopy provides a powerful approach to characterizing the mechanisms of particle motion in living cells and in other systems. We present here the theoretical basis for the analysis of these trajectories for particles undergoing random diffusion and/or systematic transport at uniform velocity in two-dimensional systems. The single particle tracking method, based on observations of the trajectories of individual particles, is compared with methods that characterize the motions of a large collection of particles such as fluorescence photobleaching recovery. Determination of diffusion coefficients or transport velocities either from correlation of positions or of velocities of the particles is discussed. A result of practical importance is an analysis of the dependence of the expected statistical uncertainty of these determinations on the number of position measurements. This provides a way of judging the accuracy of the diffusion coefficients and transport velocities obtained using this approach.

Diffusion↗
PubMed1991

Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella.

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.

Animals↗
PubMed1991

Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP.

The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db-cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl-phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A-kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillary-sized pores.

Neutrophil sequestration in pulmonary capillaries occurs prior to the development of lung injury, but the mechanisms by which neutrophils are retained are unclear. We hypothesized that decreases in cell deformability, in the absence of an increase in cell surface adhesive properties, would be sufficient to cause cell retention in a filtration apparatus modeling the pulmonary microvasculature. The myelomonocytic cell line (HL60 cell line) was used to test the hypothesis since these cells were unable to increase adherence in response to n-formylmethionylleucylphenylalanine (FMLP) in either the undifferentiated state or when differentiated towards granulocytes. With differentiation, HL60 cell volume decreased, and f-actin organization changed from a thick cortical rim with focal areas of f-actin in undifferentiated cells to a thin rim in differentiated cells. Differentiated cells responded to FMLP by reorganizing f-actin and increasing stiffness. Undifferentiated cells did not exhibit changes in f-actin with stimulation, were stiffer than differentiated cells, and did not increase stiffness in response to FMLP. Cytochalasin D (CD), which disrupted the cytoarchitecture as assessed by confocal microscopy but did not affect cell volume or adherence, decreased the stiffness of undifferentiated and FMLP-stimulated differentiated cells, thus suggesting the importance of microfilament organization in the stiffness of these cells. Filtration of cells through 8-microns pores showed that undifferentiated cells were markedly retained and did not exhibit any further retention with FMLP. Differentiated cells exposed to FMLP exhibited a concentration-dependent increase in retention in 8-microns pores that was abolished by CD. In addition, CD reduced retention of undifferentiated cells, indicating that microfilament organization is an important factor in determining a cell's rheologic properties. In conclusion, FMLP-stimulated microfilament reorganization, which increased cell stiffness, was sufficient in the absence of adherence factors to cause cell retention in a filtration system. This lends support to the hypothesis that decreases in cell deformability contribute to neutrophil retention in the pulmonary microvasculature.

Actin Cytoskeleton↗
PubMed1990

On the analysis of high order moments of fluorescence fluctuations.

A simple, straightforward analysis to characterize the distribution of aggregate sizes in a reversible aggregation system at equilibrium is presented. The method, an extension of fluorescence correlation spectroscopy (FCS), is based on measurements of higher order moments of spontaneous fluctuations of fluorescence intensity emitted from a defined open region of the sample. These fluctuations indicate fluctuations of the numbers of the fluorescent molecules in the observation region. Shot noise resulting from the random character of fluorescence emission and from the photoelectric detection system is modeled as a Poisson distribution and is subtracted from the measured photon count fluctuation moments to yield the desired fluorescence fluctuation moments. This analysis can also be used to estimate the fraction of immobile fluorophores in FCS measurements.

Mathematics↗
PubMed1990

Distribution of molecular aggregation by analysis of fluctuation moments.

The fluorescence from an open volume of a solution of fluorescent molecules fluctuates as the molecules randomly diffuse into and out of the volume. The distribution of degrees of aggregation or polymerization of the fluorescent molecules can be characterized without perturbing the system by measuring either the moments or the amplitude distribution of these fluctuations. We present an experimental verification of this approach applied to simple model systems consisting of solutions of fluorescent particles of well-defined size. We have also characterized the response of the photon-detection device (typically a photomultiplier), which is essential to the analysis of the fluorescence fluctuations, and have compared two methods for determining shot-noise contributions.

Biophysics↗
PubMed1990

Cell migration does not produce membrane flow.

We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 +/- 0.15 micron/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 +/- 2.0] x 10(-10) cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 +/- 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid.

Animals↗
PubMed1990

Mechanics of fibroblast locomotion: quantitative analysis of forces and motions at the leading lamellas of fibroblasts.

Shapes, motions, and forces developed in lamellipodia and ruffles at the leading edges of primary chick embryo heart fibroblasts were characterized by differential interference contrast microscopy and digital video enhancement techniques. The initial extension of the cell edge to form a thin, planar lamellipodium parallel to the substrate surface was analyzed in two dimensions with temporal and spatial resolution of 3 s and 0.2 micron, respectively. An extension begins and ends with brief, rapid acceleration and deceleration separated by a long period of nearly constant velocity in the range of 4-7 microns/min. Extensions and retractions were initiated randomly over time. As demonstrated by optical sectioning microscopy, the extended lamellipodia formed ruffles by sharply bending upward at hinge points 2-4 microns behind their tips. Surprisingly, ruffles continued to grow in length at the same average rate after bending upward. They maintained a straight shape in vertical cross section, suggesting the ruffles were mechanically stiff. The forces required to bend ruffles of these cells and of BC3H1 cells were measured by pushing a thin quartz fishpole probe against the tip of a ruffle 7-10 microns from its base either toward or away from the center of the cell. Force was determined by measuring the bending of the probe monitored by video microscopy. Typically the probe forced the ruffle to swing rigidly in an arc about an apparent hinge at is base, and ruffles rapidly, and almost completely, recovered their shape when the probe was removed. Hence, ruffles appeared to be relatively stiff and to resist bending with forces more elastic than viscous, unlike the cell body. Ruffles on both types of cells resisted bending with forces of 15-30 mudyn/microns of displacement at their tips when pushed toward or away from the cell center. The significance of the observations for mechanisms of cell locomotion is discussed.

Animals↗
PubMed1990

Determination of cellular mechanical properties by cell poking, with an application to leukocytes.

In this paper we review the cell-poking technique as an approach for investigating the mechanical properties of living cells. We first summarize the rationale for the technique and the mainly qualitative results obtained so far. Then we provide a technical description of the instrument as it is configured at present. This is followed by a discussion of the current status of analytical results available for interpreting cell-poking measurements. In the final section we apply these results to an analysis of unmodulated and modulated lymphocytes and neutrophils, and conclude that the mechanical response of these leukocytes to indentation is not consistent with simple models developed by previous investigators on the basis of micropipette-aspiration experiments.

Animals↗
PubMed1990

Retention of leukocytes in capillaries: role of cell size and deformability.

Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries. The size discrepancy between the mean diameter of circulating leukocytes (6-8 microns) and that of the pulmonary capillaries (approximately 5.5 microns) forces the cells to deform in order to transit the capillary bed. Consequently, we investigated the hypothesis that the biophysical properties of cell size and deformability determined differential leukocyte retention in the lung. Comparison of the filtration properties of human neutrophils, lymphocytes, monocytes, platelets, and erythrocytes through polycarbonate filters (5-micron pore diameter) revealed that the largest leukocytes (neutrophils and monocytes) were retained to the greatest extent and the smaller cells (lymphocytes and platelets) the least. Undifferentiated HL-60 cells, of greater diameter than their differentiated counterparts, were also retained to a greater extent, confirming that cell size was one important determinant of retention in these model capillaries. However, compared with neutrophils, which are of similar diameter, monocytes were retained to a greater extent, suggesting that monocytes might be less deformable than neutrophils. To test this hypothesis, deformability was measured directly using the cell poker. Monocytes were found to be the stiffest, neutrophils the softest, and lymphocytes intermediate. Glutaraldehyde treatment of neutrophils markedly increased their stiffness and decreased their ability to transit the pores of the filters in vitro and the pulmonary microvasculature of rabbits without changing their adhesive properties or size. These observations support the hypothesis that biophysical properties of leukocytes (size and deformability) determine in part their ability to transit the pulmonary capillaries and may determine the magnitude of their marginated pools.

Blood Platelets↗
PubMed1990

Activation of mechanical responses in leukocytes.

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.

Animals↗
PubMed1989

Capping of surface receptors and concomitant cortical tension are generated by conventional myosin.

We have investigated the role of cytoskeletal contraction in the capping of surface proteins crosslinked by concanavalin A on mutant Dictyostelium cells lacking conventional myosin. Measurements of cellular deformability to indicate the development of cortical tension show that cells of the wild-type parental strain, AX4, stiffen early during capping and relax back towards the softer resting state as the process is completed. Mutant cells lacking myosin (mhcA-) have a lower resting-state stiffness, and fail to stiffen and to cap crosslinked proteins on binding concanavalin A. Hence conventional myosin is essential both for capping and for the concomitant increase in cell stiffness. Furthermore, depletion of cellular ATP by azide causes a 'rigor' contraction in AX4 cells which makes them stiffen and become spherical. By contrast, the mhcA- cells fail to respond in these ways. These measurements of cortical tension in non-muscle cells can thus be directly correlated with the presence of conventional myosin, demonstrating that contractile tension generated by myosin can drive both a change of cell shape and the capping of crosslinked surface receptors.

Concanavalin A↗