Elliot L. Elson

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

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

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.

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 α‐actin and α‐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 β‐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.—Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Weil, J., Zimmermann, W., Dohmen, H. H., Schäfer, H., Bishopric, N., Wakatsuki, T., Elson, E. L. Three‐dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11, 683–694 (1997)

Capping protein levels influence actin assembly and cell motility in dictyostelium

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.

Determination of Cellular Mechanical Properties by Cell Poking, With an Application to Leukocytes

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

On the analysis of high order moments of fluorescence fluctuations

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

Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross‐correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.

Mechanics of cell spreading: role of myosin II

As it migrates over a substratum, a cell must exert different kinds of forces that act at various cellular locations and at specific times. These forces must therefore be coordinately regulated. The Rho-family GTPases Rac1 and Cdc42 promote actin polymerization that drives extension of the leading cell edge. Subsequently, RhoA regulates myosin-dependent contractile force, which is required for formation of adhesive contacts and stress fibers. During cell spreading, however, the activity of RhoA is reduced by a mechanism involving the tyrosine kinases c-Src and focal adhesion kinase (FAK), and the p190RhoGAP. It has been proposed that this reduction of RhoA activity facilitates edge extension by reducing myosin-dependent contractile forces that could resist this process. We have directly tested this hypothesis by correlating myosin activity with the rate of cell spreading on a substratum. The rate of spreading is inversely related to the myosin activity. Furthermore, spreading is inhibited by low concentrations of cytochalasin D, as expected for a process that depends on the growth of uncapped actin filaments. Cell indentation measurements show that a myosin-dependent viscoelastic force resists cell deformation.

Concentration fluctuations in a mesoscopic oscillating chemical reaction system.

Under sustained pumping, kinetics of macroscopic nonlinear biochemical reaction systems far from equilibrium either can be in a stationary steady state or can execute sustained oscillations about a fixed mean. For a system of two dynamic species X and Y, the concentrations nx and ny will be constant or will repetitively trace a closed loop in the (nx, ny) phase plane, respectively. We study a mesoscopic system with nx and ny very small; hence the occurrence of random fluctuations modifies the deterministic behavior and the law of mass action is replaced by a stochastic model. We show that nx and ny execute cyclic random walks in the (nx, ny) plane whether or not the deterministic kinetics for the corresponding macroscopic system represents a steady or an oscillating state. Probability distributions and correlation functions for nx(t) and ny(t) show quantitative but not qualitative differences between states that would appear as either oscillating or steady in the corresponding macroscopic systems. A diffusion-like equation for probability P(nx, ny, t) is obtained for the two-dimensional Brownian motion in the (nx, ny) phase plane. In the limit of large nx, ny, the deterministic nonlinear kinetics derived from mass action is recovered. The nature of large fluctuations in an oscillating nonequilibrium system and the conceptual difference between “thermal stochasticity” and “temporal complexity” are clarified by this analysis. This result is relevant to fluorescence correlation spectroscopy and metabolic reaction networks.

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel β-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 μsec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.

Quick tour of fluorescence correlation spectroscopy from its inception.

Fluorescence correlation spectroscopy (FCS) was originally developed in the early 1970s as a way to measure the kinetics of chemical reactions under zero perturbation conditions. At its inception, the measurement was difficult due to experimental limitations and was primarily used during the 1970s and 1980s to characterize diffusion. More recently, as a result of technological advances, FCS measurements have become easier and more versatile. In addition to measurements of diffusion both in solution and in cells, FCS is now also used to measure not only chemical reaction kinetics but also extents of molecular aggregation, the dynamics of photophysical processes, conformational fluctuations, molecular interactions in solution and in cells, and has even found application as a pharmaceutical screening method. From its inception to the present, the contributions of Webb and his coworkers have had a central and defining role in the development and applications of FCS.