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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nanometre-level analyses of the movements of membrane glycoproteins tagged with gold particles demonstrate that diffusing particles are not under the influence of a lipid flow, although a subset of particles which appear attached to the cytoskeleton are moving rearward.

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.

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.

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).

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.

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.

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.

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.

Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10(-9) cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10(-10)-10(-11) cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodamine-phosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltage-dependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.

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.

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.

A review with 57 refs.

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.

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.

Cross-linking surface immunoglobulin (sIg) on B-lymphocytes causes a substantial increase in the mechanical stiffness of the cells. This has been demonstrated using a new method for measuring cellular deformability. The method is based on a device, the "Cell Poker", which we use to determine the force required slightly to indent or compress a cell adherent to a rigid substrate in culture. Cross-linking of sIg by bivalent anti-sIg antibodies is necessary to elicit the increase in stiffness; binding of monovalent Fab fragments is insufficient. The increase in stiffness is partially reversed by cytochalasin D and by completion of the capping of the cross-linked sIg. The modulation of cellular deformability and the induction of cellular dynamic processes such as capping are similar in their requirements for cross-linking sIg and in their sensitivity to cytochalasins. This suggests that both kinds of responses stem from similar cellular processes and structures. These results emphasize the mechanical capability of lymphocytes and suggest that the physiological functions of these cells are likely to employ this capability.

Con A crosslinking to lymphocyte surface glycoproteins resulted in increased cytoplasmic viscoelasticity and resistance to cell deformation (stiffness). Actin microfilaments were involved, since cytochalasin D treatment reversed the effects of Con A crosslinking. Surface acceptor crosslinking apparently alters the mech. state of the cytoskeleton by limiting the mobility of cytoskeleton proteins. The changes in cytoplasmic viscoelasticity also demonstrate signal transfer from cell modification.

Cell poking, a new method for measuring mechanical properties of single cells was used to determine the elastic area compressibility modulus of osmotically swollen human erythrocytes. With this method we determined the force required to indent cells attached to a glass coverslip (Petersen, N.O., W. B. McConnaughey , and E. L. Elson , 1982, Proc. Natl. Acad. Sci. USA, 79:5327. Forces on the order of one millidyne and indentations on the order of one micron were detected. An analysis of these data in terms of a simplified mechanical model yielded the elastic area compressibility modulus. This analysis used a variational approach to minimize the isothermal elastic potential energy density function given by E. A. Evans and R. Skalak (Mechanics and Thermodynamics of Biomembranes, 1980, CRC Press, Boca Raton , FL). Measurements on swollen erythrocytes gave a range of values, depending in part on the osmotic conditions, of 17.9 +/- 8.2 to 34.8 +/- 12.0 mdyn /micron for the elastic area compressibility modulus at 25 degrees C. Fractional area expansion greater than 2.6 +/- 0.8% produced rapid cell lysis. These values were not corrected for the reversible movement of water across the cell membrane in response to hydrostatic pressure gradients. Our results agree reasonably with those obtained by Evans et al. (Evans, E.A., R. Waugh , and L. Melnick , 1976, Biophys. J., 16:585-595.) using micropipette aspiration under similar conditions.

Ethidium bromide binding to DNA was studied at different DNA concns., and a single-site model gave results identical to those given by a multiple-site model for fluorescence photobleaching recovery (FPR) measurements. This is in contrast to the expected and obsd. behavior of fluorescence correlation spectroscopy (FCS) measurements. The FCS and FPR measurements also reveal an apparently enhanced diffusion of ethidium at high DNA concn. This enhancement might result from direct transfer of ethidium among DNA mols.

Fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) were applied to systems in which a fluorescent ligand can bind to a polyvalent substrate mol. in a multistep reaction sequence. The anal. theory for both FCS and FPR is extended to allow anal. of these kinds of systems. Exptl. measurements of the binding of ethidium bromide to DNA by FCS confirm the theor. anal. The anal. shows that FCS and FPR perceive multivalent binding reactions differently. This difference results from the selective effect of the photobleaching process in the chem. reaction system. The development and results have useful applications to a wide range of biopolymeric binding and assembly processes.