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Here are papers from the lab over the last several years. We also offer older papers and some really old papers. 1. Effects of cytochalasin D and latrunculin B on mechanical properties of
cells. Wakatsuki, T., B. Schwab, N.C. Thompson, and E.L. Elson Actin microfilaments transmit traction and contraction forces generated within a cell to the extracellular matrix during embryonic development, wound healing and cell motility, and to maintain tissue structure and tone. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and tissues. Cytochalasin D and Latrunculin are commonly used reagents that, by different mechanisms, alter the state of actin polymerization or the organization of actin filaments. We have investigated the effect of a wide range of Cytochalasin D and Latrunculin B concentrations (from 40 pM to 10 microM) on the mechanical properties of the cells within fibroblast populated collagen matrices. Contractile force and dynamic stiffness were measured by uniaxial stress-strain testing. The range of effective concentrations of Cytochalasin D (200 pM-2 microM) was broader than that of Latrunculin B (20 nM-200 nM). Activating the cells by serum did not change the effective range of Cytochalasin D concentrations but shifted that of Latrunculin B upward by tenfold. Simple mathematical binding models based on the presumed mechanisms of action of Cytochalasin D and Latrunculin B simulated the concentration-dependent mechanical changes reasonably well. This study shows a strong dependence of the mechanical properties of cells and tissues on the organization and degree of polymerization of actin filaments. 2. Substrate recognition by gelatinase a: the c-terminal domain
facilitates surface diffusion. Collier, I.E., S. Saffarian, B.L. Marmer, E.L.
Elson, and G. Goldberg An investigation of gelatinase A binding to gelatin produced results that are inconsistent with a traditional bimolecular Michaelis-Menten formalism but are effectively accounted for by a power law characteristic of fractal kinetics. The main reason for this inconsistency is that the bulk of the gelatinase A binding depends on its ability to diffuse laterally on the gelatin surface. Most interestingly, we show that the anomalous lateral diffusion and, consequently, the binding to gelatin is greatly facilitated by the C- terminal hemopexin-like domain of the enzyme whereas the specificity of binding resides with the fibronectin-like gelatin-binding domain. 3. A cell-based constitutive relation for bio-artificial tissues.
Zahalak, G.I., J.E. Wagenseil, T. Wakatsuki, and E.L. Elson By using a combination of continuum and statistical mechanics we derive an integral constitutive relation for bio-artificial tissue models consisting of a monodisperse population of cells in a uniform collagenous matrix. This constitutive relation quantitatively models the dependence of tissue stress on deformation history, and makes explicit the separate contribution of cells and matrix to the mechanical behavior of the composite tissue. Thus microscopic cell mechanical properties can be deduced via this theory from measurements of macroscopic tissue properties. A central feature of the constitutive relation is the appearance of "anisotropy tensors" that embody the effects of cell orientation on tissue mechanics. The theory assumes that the tissues are stable over the observation time, and does not in its present form allow for cell migration, reorientation, or internal remodeling. We have compared the predictions of the theory to uniaxial relaxation tests on fibroblast-populated collagen matrices (FPMs) and find that the experimental results generally support the theory and yield values of fibroblast contractile force and stiffness roughly an order of magnitude smaller than, and viscosity comparable to, the corresponding properties of active skeletal muscle. The method used here to derive the tissue constitutive equation permits more sophisticated cell models to be used in developing more accurate representations of tissue properties. 4. Cell mechanics studied by a reconstituted model tissue. Wakatsuki,
T., M.S. Kolodney, G.I. Zahalak, and E.L. Elson Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells. 5. Collagen receptor control of epithelial morphogenesis and cell cycle
progression. Zutter, M.M., S.A. Santoro, J.E. Wu, T. Wakatsuki, S.K.
Dickeson, and E.L. Elson To define the unique contributions of the alpha subunit cytoplasmic tails of the alpha(1)beta(1) and alpha(2)beta(1) integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking alpha(1)beta(1) and alpha(2)beta(1) integrin expression was stably transfected with the full-length alpha(2) integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the alpha(2) subunit and the cytoplasmic domain of the alpha(1) subunit (X2C1), or alpha(2) cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three- dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the alpha(2) integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact alpha(1) or alpha(2) integrin subunit cytoplasmic domain can promote cell cycle progression. 6. Quantitative study of polymer conformation and dynamics by single-
particle tracking. Qian, H. and E.L. Elson We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single- particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level. 7. Weak dependence of mobility of membrane protein aggregates on aggregate
size supports a viscous model of retardation of diffusion. Kucik, D.F., E.L.
Elson, and M.P. Sheetz Proteins in plasma membranes diffuse more slowly than proteins inserted into artificial lipid bilayers. On a long-range scale (>250 nm), submembrane barriers, or skeleton fences that hinder long-range diffusion and create confinement zones, have been described. Even within such confinement zones, however, diffusion of proteins is much slower than predicted by the viscosity of the lipid. The cause of this slowing of diffusion on the micro scale has not been determined and is the focus of this paper. One way to approach this question is to determine the dependence of particle motion on particle size. Some current models predict that the diffusion coefficient of a membrane protein aggregate will depend strongly on its size, while others do not. We have measured the diffusion coefficients of membrane glycoprotein aggregates linked together by concanavalin A molecules bound to beads of various sizes, and also the diffusion coefficients of individual concanavalin A binding proteins. The measurements demonstrate at most a weak dependence of diffusion coefficient on aggregate size. This finding supports retardation by viscous effects, and is not consistent with models involving direct interaction of diffusing proteins with cytoskeletal elements. 8. Forces in cell locomotion. Elson, E.L., S.F. Felder, P.Y. Jay, M.S.
Kolodney, and C. Pasternak The molecular mechanisms that drive animal cell locomotion are partially characterized, but not definitively understood. It seems likely that actin polymerization contributes to the forward protrusion of the leading edge of a migrating cell. Both myosin-dependent contractile forces and selective detachment of adhesive interactions with the substratum seem to contribute to release of the posterior of an extended cell. It is probable, but not certain, that a separate 'traction' force advances the cell body towards the forward anchorage sites formed by the advancing lamellipodium. The molecular mechanism of this force is unknown. Determining the role of traction forces in migrating fibroblasts and keratocytes is complicated by the fact that the primary functions of the relatively strong forces exerted on the substratum by these cells may be to establish tissue 'tone' and to remodel tissue matrices, rather than to drive locomotion. In accordance with this notion, rapidly moving cells such as neutrophils and Dictyostelium amoebae exert weaker forces on the substratum as they migrate. The traction force in cell migration may be distinct from traction forces with tissue functions. Ultimately, the mechanism may be revealed by using molecular genetics to disrupt the motors that provide this force. Reconstituted tissues provide systems in which to investigate the regulation of cell forces and their contribution to tissue mechanical properties and development. 9. Axial and transverse stiffness measures of cochlear outer hair cells
suggest a common mechanical basis. Ulfendahl, M., E. Chan, W.B. McConnaughey,
S. Prost-Domasky, and E.L. Elson The function of the hearing organ is based on mechanical processes occurring at the cellular level. The mechanical properties of guinea- pig isolated sensory cells were investigated using two different techniques. The stiffness of the outer hair cells along the longitudinal axis was measured by compressing the cell body using stiffness-calibrated quartz fibres. For cells with a mean length of 69 micron, the mean axial compression stiffness was 1. 1+/-0.8 mN/m (+/- SD). There was an inverse relation between stiffness and cell length. The stiffness of the cell membrane perpendicular to the longitudinal axis of the sensory cell was measured by indenting the cell membrane with a known force. The mean lateral indentation stiffness was 3.3+/- 1.5 mN/m (+/-SD) for cells with a mean length of 64 microm. Longer cells were less stiff than short cells. Modelling the hair cell as a shell with bending resistance, finite element calculations demonstrated that the axial compression stiffness correlated well with the lateral indentation stiffness, and that a simple isotropic model is sufficient to explain the experimental observations despite the different stress strain states produced by the two techniques. The results imply that the two different stiffness properties may originate from the same cytoskeletal structures. It is suggested that the mechanical properties of the outer hair cells are designed to influence the sound-induced motion of the reticular lamina. In such a system, stiffness changes of the outer hair cell bodies could actively control the efficiency of the mechanical coupling between the basilar membrane and the important mechanoelectrical transduction sites at the surface of the hearing organ. 10. Role of ponticulin in pseudopod dynamics, cell-cell adhesion, and
mechanical stability of an amoeboid membrane skeleton. Luna, E.J., A.L. Hitt,
D. Shutt, D. Wessels, D. Soll, P. Jay, C. Hug, E.L. Elson, A. Vesley, G.P.
Downey, M. Wang, S.M. Block, W. Sigurdson, and F. Sachs 11. Three-dimensional reconstitution of embryonic cardiomyocytes in a
collagen matrix: a new heart muscle model system. Eschenhagen, T., C. Fink,
U. Remmers, H. Scholz, J. Wattchow, J. Weil, W. Zimmermann, H.H. Dohmen, H.
Schafer, N. Bishopric, T. Wakatsuki, and E.L. Elson A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha- tropomyosin-positive cardiac myocytes exhibiting typical cross- striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive "staircase"), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions. 12. Expression of the catalytic domain of myosin light chain kinase
increases paracellular permeability. Hecht, G., L. Pestic, G. Nikcevic, A.
Koutsouris, J. Tripuraneni, D.D. Lorimer, G. Nowak, V. Guerriero, Jr., E.L.
Elson, and P.D. Lanerolle Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin- Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta- gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability. 13. Mechanical function of dystrophin in muscle cells. Pasternak, C.,
S. Wong, and E.L. Elson We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells. 14. Fibroblast contractility without an increase in basal myosin light
chain phosphorylation in wild type cells and cells expressing the catalytic
domain of myosin light chain kinase. Obara, K., G. Nikcevic, L. Pestic, G.
Nowak, D.D. Lorimer, V. Guerriero, Jr., E.L. Elson, R.J. Paul, and P. de
Lanerolle We investigated the role of myosin light chain (MLC20) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC- P is 0.96 MOL of PO4/mol of MOL20 in cell expressing tMK compared to 0.20 mol of PO4/mol of MLC20 in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers. 15. Contraction due to microtubule disruption is associated with increased
phosphorylation of myosin regulatory light chain. Kolodney, M.S. and E.L.
Elson Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC20). We measured the force produced by a population of fibroblasts within a collagen lattice attached to an isometric force transducer. Treatment of cells with nocodazole, an inhibitor of microtubule polymerization, stimulated an isometric contraction that reached its peak level within 30 min and was typically 30-45% of the force increase following maximal stimulation with 30% fetal bovine serum. The contraction following nocodazole treatment was associated with a 2- to 4-fold increase in LC20 phosphorylation. The increases in both force and LC20 phosphorylation, after addition of nocodazole, could be blocked or reversed by stabilizing the microtubules with paclitaxel (former generic name, taxol). Increasing force and LC20 phosphorylation by pretreatment with fetal bovine serum decreased the subsequent additional contraction upon microtubule disruption, a finding that appears inconsistent with a load-shifting mechanism. Our results suggest that phosphorylation of LC20 is a common mechanism for the contractions stimulated both by microtubule poisons and receptor- mediated agonists. The modulation of myosin activity by alterations in microtubule assembly may coordinate the physiological functions of these cytoskeletal components. 16. A mechanical function of myosin II in cell motility. Jay, P.Y.,
P.A. Pham, S.A. Wong, and E.L. Elson Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell's rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild- type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface. 17. Capping protein levels influence actin assembly and cell motility in
dictyostelium. Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L.
Elson, and J.A. Cooper Actin assembly is important for cell motility, but the mechanism of assembly and how it relates to motility in vivo is largely unknown. In vitro, actin assembly can be controlled by proteins, such as capping protein, that bind filament ends. To investigate the function of actin assembly in vivo, we altered the levels of capping protein in Dictyostelium cells and found changes in resting and chemoattractant- induced actin assembly that were consistent with the in vitro properties of capping protein in capping but not nucleation. Significantly, overexpressers moved faster and underexpressers moved slower than control cells. Mutants also exhibited changes in cytoskeleton architecture. These results provide insights into in vivo actin assembly and the role of the actin cytoskeleton in motility. 18. Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. Kolodney, M.S. and E.L. Elson J. Biol. Chem., 1993. 268(32), 23850-5. In vitro studies have indicated that the enzymatic activity of myosin II from non-muscle cells is controlled by phosphorylation of its regulatory light chain (LC20). We have studied one likely functional consequence of phosphorylating LC20 in living chick embryo fibroblasts (CEF) by measuring contractile force developed by these cells. Using a recently developed method, we recorded quantitative changes in isometric force generated by a population of cells following mitogenic stimulation. Fetal bovine serum, thrombin, and lysophosphatidic acid stimulate rapid isometric contraction of CEF. Cells stimulated with thrombin develop maximal force within 5-10 min. Force development correlates temporally with a 3-5-fold increase in the overall fraction of LC20 phosphorylated and with the fractions of LC20 in both the monophosphorylated and diphosphorylated states. Unloaded shortening velocity also increases after thrombin stimulation. Although both force and phosphorylation begin to decline 10 min after stimulation, the level of phosphorylation declined more rapidly than the force. These results suggest that the role of LC20 phosphorylation in regulating fibroblast contractility is analogous to its well established role in regulating smooth muscle contraction and that quantitative measurements of the force developed by populations of fibroblasts (or other cultured cells) can be used to study the regulation of non-sarcomeric myosin at the molecular level in vivo. 19. Studies of mechanical aspects of amoeboid locomotion. Jay, P.Y.,
C. Pasternak, and E.L. Elson When a cell crawls over a surface, it exerts forces which both change its shape and deformability and propel it forward. The mechanisms involved are poorly understood. They can best be studied by combining biochemical and molecular genetic methods with direct, quantitative measurements of mechanical properties. Measurements of cellular deformability provide indications of contractile tension developed within the cell and of cytoskeletal reorganizations which influence local cellular viscoelasticity. An example is the capping of cross- linked cell surface proteins, which occurs on cells as diverse as mammalian lymphocytes and the unicellular amoeba, Dictyostelium discoideum. Deformability measurements show that cells stiffen as they cap. Measurements on wild-type Dictyostelium cells and on cells engineered to lack conventional myosin (myosin II) demonstrate that capping requires myosin II and that the concurrent cellular stiffening results from a myosin-II-dependent contractile force. Measurements of the systematic transport of beads rearward over the surfaces of cells characterize a mechanism of movement which could be used to drive the cell forward. Capping is one such mechanism. A distinct myosin-II- independent form of rearward transport is revealed in measurements of fluorescent beads on the Dictyostelium cells which lack this protein. In addition to studies of cell locomotion, measurements of cellular mechanical properties can provide quantitative assays of the functions of cytoskeletal components. Such studies are motivated by the nature of cytoskeletal proteins whose function, in contrast to enzymes, are mechanical rather that catalytic. 20. Studies on the structure of actin gels using time correlation
spectroscopy of fluorescent beads. Qian, H., E.L. Elson, and C. Frieden 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). 21. Kinetics and mechanism of the folding of cytochrome c. Pryse, K.M., T.G. Bruckman, B.W. Maxfield, and E.L. Elson Biochemistry, 1992. 31(22), 5127-36. 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. 22. Surface particle transport mechanism independent of myosin II in Dictyostelium. Jay, P.Y. and E.L. Elson Nature, 1992. 356(6368), 438-40. 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. 23. Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties. Erzurum, S.C., G.P. Downey, D.E. Doherty, B. Schwab, 3rd, E.L. Elson, and G.S. Worthen J. Immunol., 1992. 149(1), 154-62. 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. 24. Single particle tracking. Analysis of diffusion and flow in two- dimensional systems. Qian, H., M.P. Sheetz, and E.L. Elson Biophys. J., 1991. 60(4), 910-21. 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. 25. Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella. Kucik, D.F., S.C. Kuo, E.L. Elson, and M.P. Sheetz J. Cell Biol., 1991. 114(5), 1029-36. 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. 26. Actin tracks. Kucik, D.F., E.L. Elson, and Y.L. Wang Nature, 1991. 354(6352), 362-3. 27. Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillary-sized pores. Erzurum, S.C., M.L. Kus, C. Bohse, E.L. Elson, and G.S. Worthen Am. J. Respir. Cell Mol. Biol., 1991. 5(3), 230-41. 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. 28. Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP. Downey, G.P., E.L. Elson, B. Schwab, 3rd, S.C. Erzurum, S.K. Young, and G.S. Worthen J. Cell Biol., 1991. 114(6), 1179-90. 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. 29. Determination of cellular mechanical properties by cell poking, with an application to leukocytes. Zahalak, G.I., W.B. McConnaughey, and E.L. Elson J. Biomech. Eng., 1990. 112(3), 283-94. 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. 30. On the analysis of high order moments of fluorescence fluctuations. Qian, H. and E.L. Elson Biophys. J., 1990. 57(2), 375-80. 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. 31. Distribution of molecular aggregation by analysis of fluctuation moments. Qian, H. and E.L. Elson Proc. Natl. Acad. Sci. USA, 1990. 87(14), 5479-83. 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. 32. Cell migration does not produce membrane flow. Kucik, D.F., E.L. Elson, and M.P. Sheetz J. Cell Biol., 1990. 111(4), 1617-22. 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. 33. Mechanics of fibroblast locomotion: quantitative analysis of forces and motions at the leading lamellas of fibroblasts. Felder, S. and E.L. Elson J. Cell Biol., 1990. 111(6 Pt 1), 2513-26. 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. 34. Activation of mechanical responses in leukocytes. Elson, E.L., C. Pasternak, Z.Y. Liu, J.I. Young, B. Schwab, 3rd, G.S. Worthen, G. Downey, R. Michaels, W.B. McConnaughey, M. McDaniel, and et al. Biorheology, 1990. 27(6), 849-58 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. 35. Retention of leukocytes in capillaries: role of cell size and deformability. Downey, G.P., D.E. Doherty, B. Schwab, 3rd, E.L. Elson, P.M. Henson, and G.S. Worthen J. Appl. Physiol., 1990. 69(5), 1767-78. 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. |