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Biomedical subjects

E L Elson

Publications and source records attributed to E L Elson.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

Forward transport of glycoproteins on leading lamellipodia in locomoting cells.

In several types of locomoting cells, active rearward transport of particles on the cell surface has been observed and correlated with motility. No forward transport of particles has previously been reported, however. Here we report rapid forward transport of concanavalin A-coated gold particles on the dorsal surfaces of lamellipodia of fish epidermal keratocytes. These movements are active, not diffusive, and more rapid than either rearward particle transport or the rate of cell locomotion. We observed forward transport in migrating, but not in stationary cells, and could block the movement by treatment with cytochalasin D. These studies demonstrate for the first time that small numbers of glycoproteins can be actively transported on the surface of the cell to the front of the lamellipodium. We suggest that this mechanism transports proteins involved in cell locomotion, such as proteins necessary for adhesion, and could also produce an extensile force.

Animals↗
PubMed1989

Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries.

The effect of peptide chemoattractants on neutrophil mechanical properties was studied to test the hypothesis that stimulated neutrophils (diameter, 8 micrometers) are retained in pulmonary capillaries (5.5 micrometers) as a result of a decreased ability of the cell to deform within the capillary in response to the hydrodynamic forces of the bloodstream. Increased neutrophil stiffness, actin assembly, and retention in both 5-micrometer pores and the pulmonary vasculature were seen in response to N-formyl-methionyl-leucyl-phenylalanine. These changes were abolished in cells that had been incubated with 2 micromolar cytochalasin D, an agent that disrupts cellular actin organization. A monoclonal antibody directed at the CD11-CD18 adhesive glycoprotein complex did not inhibit the increase in stiffness or retention in pores. These data suggest that neutrophil stiffening may be both necessary and sufficient for the retention that is observed. Hence, neutrophil sequestration in lung and other capillaries in the acute inflammatory process may be the result of increased stiffness stimulated by chemoattractants.

Actin Cytoskeleton↗
PubMed1989

Cell poking: quantitative analysis of indentation of thick viscoelastic layers.

A recently introduced device, the cell poker, measures the force required to indent the exposed surface of a cell adherent to a rigid substratum. The cell poker has provided phenomenological information about the viscoelastic properties of several different types of cells, about mechanical changes triggered by external stimuli, and about the role of the cytoskeleton in these mechanical functions. Except in special cases, however, it has not been possible to extract quantitative estimates of viscosity and elasticity moduli from cell poker measurements. This paper presents cell poker measurements of well characterized viscoelastic polymeric materials, polydimethylsiloxanes of different degrees of polymerization, in a simple shape, a flat, thick layer, which for our purposes can be treated as a half space. Analysis of the measurements in terms of a linear viscoelasticity theory yields viscosity values for three polymer samples in agreement with those determined by measurements on a macroscopic scale. Theoretical analysis further indicates that the measured limiting static elasticity of the layers may result from the tension generated at the interface between the polymer and water. This work demonstrates the possibility of obtaining quantitative viscoelastic material properties from cell poker measurements and represents the first step in extending these quantitative studies to more complicated structures including cells.

Cell Adhesion↗
PubMed1989

Interpretation of fluorescence correlation spectroscopy and photobleaching recovery in terms of molecular interactions.

The theoretical basis and experimental implementation of FCS and FPR measurements are now well established. Because of the requirements for system stability and long data acquisition times FCS is relatively rarely used. But FCS can provide unique information, especially about extents of aggregation or polymerization and therefore is a useful supplement to FPR for certain applications. FPR measurements are now carried out routinely in many laboratories in a variety of formats using different beam profiles, optical systems, and analytical schemes. A particular version may be better adapted to a specific application. The spot photobleaching approach, however, seems simplest and most versatile for cellular studies and is now most often used. Important experimental considerations in setting up a spot photobleaching instrument are discussed in detail in Chapter 10 by Wolf (this volume) and elsewhere (Petersen et al., 1986a). In interpreting FPR measurements it is also important to take into account the possibility of systematic errors from a number of sources. In Chapter 10 in this volume, Wolf discusses many factors that must be properly controlled in carrying out FPR measurements. Additional consideration of some of these points is presented by Petersen et al. (1986a). One potentially troublesome type of error arises from the possibility that chemical reactions initiated by the photobleaching pulse or during the measurement of recovery could significantly perturb the system. Evidence from a variety of sources [summarized, for example, in Petersen et al. (1986a)] indicates that photobleaching fluorophores can induce chemical cross-linking of cellular proteins under some conditions. But measurements in a number of different systems have demonstrated that, even if these types of reactions occur in FPR measurements, nevertheless they do not perturb the measured mobilities. If possible, however, this point should be checked for each new system because variations in structure or environmental conditions could enhance the chemical cross-linking reactions mediated by photogenerated free radicals. In practice, the principal difficulty in carrying out FPR measurements on cells is frequently the low intensity of the fluorescent signal which can be obtained from specifically labeled cell surface ligands or microinjected components. This low intensity results from the typically low capacity of an individual cell for the specifically labeled macromolecule. Even in the absence of systematic errors, low emission intensity will reduce the accuracy of measurements due to shot noise. This is an important practical limitation on measuring accuracy. Low measurement accuracy severely limits the extent to which the data can be interpreted mechanistically. Precision can be improved by averaging many recovery experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Diffusion↗
PubMed1989

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.

Actins↗
PubMed1989

Formation of acetylcholine receptor clusters in chick myotubes: migration or new insertion?

Experiments were performed to study the feasibility of two mechanisms of acetylcholine receptor (ACHR) accumulation in chick myotubes: diffusion and trapping of previously dispersed surface receptors and localized insertion of new receptors at accumulation sites. Fluorescence photobleaching recovery (FPR) measurements indicated that the majority of diffusely distributed ACHRs in chick myotube membranes were mobile whereas nearly all receptors within high density clusters were effectively immobile. Unlike previous reports, two rates of ACHR movement characterized the mobile population. Moreover, we found that the estimated diffusion coefficient depended critically on the objective (spot size) used to assay recovery from bleaching. Implications of this finding for mechanisms of receptor immobilization are discussed. Extracts of chick brain, known to increase the number of surface receptors, did not alter receptor mobility. Extracts of Torpedo electric organ that increase the number of receptor aggregates, decreased the mobile fraction of ACHRs. Simulations of the diffusion and trapping mechanism indicated that captured receptors should congregate around the periphery of a receptor patch during the first hour after they were inserted into the membrane. However, newly inserted ACHRs were found to be located centrally within receptor patches under neurites, and this was not consistent with an exclusive diffusion-trapping mechanism. We also studied the mobility of ACHRs near points of contact made by cholinergic growth cones. The rate of receptor movement was increased in the vicinity of growth cones, but the magnitude of this effect was small.

Animals↗
PubMed1988

Localization and mobility of gelsolin in cells.

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.

Animals↗
PubMed1987

Microinjection of gelsolin into living cells.

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

Animals↗
PubMed1987

Rat basophilic leukemia cells stiffen when they secrete.

RBL cells provide a useful model of the IgE and antigen-dependent stimulus-secretion coupling of mast cells and basophils. We have measured cellular deformability to investigate the participation of cytoskeletal mechanical changes. Cross-linking cell-surface IgE-receptor complexes with multivalent ligands not only triggered secretion but also caused the cells to stiffen, i.e., to become more resistant to deformation. This mechanical response required receptor cross-linking, had a time course similar to that of secretion, and was reversed by DNP-L-lysine, a competitive inhibitor of antigen binding. Hence the same stimulus seems to elicit both stiffening and secretion. Cytochalasin D, which inhibits actin filament assembly, prevented or reversed stiffening, thereby implicating the cytoskeleton in the mechanical response. Increasing intracellular calcium ion concentration with the ionophore A23187 stiffened cells and stimulated secretion. Activation of protein kinase C with a phorbol ester also stiffened cells and enhanced both the stiffening and secretion caused by the ionophore. Yet cytochalasin D enhances secretion whereas activation of protein kinase c alone is insufficient for secretion. Therefore stiffening is neither necessary nor sufficient for secretion. These results characterize a cytoskeletal mechanical response triggered by the same receptor-dependent stimulus that elicits secretion and by second messengers that are thought to mediate between the receptor signal and secretion. The function of the mechanical response, however, remains to be determined.

Animals↗
PubMed1986

Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2.

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.

Aminoquinolines↗
PubMed1985

Lymphocyte mechanical response triggered by cross-linking surface receptors.

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.

Animals↗