Yi-Der Chen

A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin

Binding of caldesmon to actin causes a decrease in the quantity of bound myosin and results in a reduction in the rate of actin-activated adenosine triphosphate hydrolysis. It is generally assumed that the binding of caldesmon and myosin to actin is a pure competitive interaction. However, recent binding studies of enzyme digested caldesmon subfragments directed at mapping the actin binding site of caldesmon have shown that a small 8-kD fragment around the COOH-terminal can compete directly with the myosin subfragment 1 (S-1) binding to actin; at least one other fragment that binds to actin does not inhibit the actin-activated adenosine triphosphate activity of myosin. That is, only a part of the caldesmon sequence may be responsible for directly blocking the binding of S-1 to actin. This prompts us to question the actual mode of binding of intact caldesmon and myosin S-1 to actin: whether the entire intact caldesmon molecule is competing with S-1 binding (pure competitive model) or just a small part of it (mosaic multiple-binding model). To answer this question, we measured the amount of myosin S-1 and caldesmon bound per actin monomer as a function of the total concentration of S-1 added to the system at constant concentrations of actin and caldesmon. A formalism for calculating the titration data based on the pure competitive model and a mosaic multiple-binding model was then developed. When compared with theoretical calculations, it is found that the binding of caldesmon and S-1 to actin cannot be pure competitive if no cooperativity exists between S-1 and caldesmon. In contrast, the mosaic multiple-binding model can fit the binding data rather well regardless of the existence of cooperativity between S-1 and caldesmon.

Fluorescence dequenching kinetics of single cell-cell fusion complexes

In an earlier paper which models the cell-cell (or virus-cell) fusion complex as two partial spherical vesicles joined at a narrow neck (Rubin, R. J., and Yi-der Chen. 1990. Biophys. J. 58:1157-1167), the redistribution by diffusion of lipid-like molecules through the neck between the two fused cell surfaces was studied. In this paper, we extend the study to the calculation of the kinetics of fluorescence increase in a single fusion complex when the lipid-like molecules are fluorescent and self-quenching. The formalism developed in this paper is useful in deducing fusion activation mechanisms from cuvette fluorescence measurements in cell-cell fusion systems. Two different procedures are presented: 1) an exact one which is based on the exact local density functions obtained from diffusion equations in our earlier study; and 2) an approximate one which is based on treating the kinetics of transfer of probes between the two fused cells as a two-state chemical reaction. For typical cell-cell fusion complexes, the fluorescence dequencing curves calculated from the exact and approximate procedures are very similar. Due to its simplicity, the approximate method should be very useful in future applications. The formalism is applied to a typical cell-cell fusion complex to study the sensitivity of dequenching curves to changes in various fusion parameters, such as the radii of the cells, the radius of the pore at the fusion junction, and the number of probes initially loaded to the complex.

Fluctuations and Randomness of Movement of the Bead Powered by a Single Kinesin Molecule in a Force-Clamped Motility Assay:Monte Carlo Simulations

The motility assay of K. Visscher, M. J. Schnitzer, and S. M. Block (Nature, 400:184-189, 1999) in which the movement of a bead powered by a single kinesin motor can be measured is a very useful tool in characterizing the force-dependent steps of the mechanochemical cycle of kinesin motors, because in this assay the external force applied to the bead can be controlled (clamped) arbitrarily. However, because the bead is elastically attached to the motor and the response of the clamp is not fast enough to compensate the Brownian motion of the bead, interpretation or analysis of the data obtained from the assay is not trivial. In a recent paper (Y. Chen and B. Yan, Biophys. Chem. 91:79-91, 2001), we showed how to evaluate the mean velocity of the bead and the motor in the motility assay for a given mechanochemical cycle. In this paper we extend the study to the evaluation of the fluctuation or the randomness of the velocity using a Monte Carlo simulation method. Similar to the mean, we found that the randomness of the velocity of the motor is also influenced by the parameters that affect the dynamic behavior of the bead, such as the viscosity of the medium, the size of the bead, the stiffness of the elastic element connecting the bead and the motor, etc. The method presented in this paper should be useful in modeling the kinetic mechanism of any processive motor (such as conventional kinesin and myosin V) based on measured force-clamp motility data.

Diffusion and redistribution of lipid-like molecules between membranes in virus-cell and cell-cell fusion systems

The kinetics of redistribution of lipid-like molecules between the membranes of two fused spherical vesicles is studied by solving the time-dependent diffusion equation of the system. The effects on the probe redistribution rate of pore size at the fusion junction and the relative sizes of the vesicles are examined. It is found that the redistribution rate constant decreases significantly, but not drastically, as the relative size of the pore to that of the vesicles decreases (the bottleneck effect). In general, the time scale of the probe redistribution rate is determined by the size of the vesicles that is loaded with the probe before the activation of the fusion. For a pore size 50 A in diameter and a typical diffusion coefficient of 10(-8) cm2/s for lipids, the mixing half times for typical virus-cell and cell-cell fusion systems are less than 30 ms and above 200 s, respectively. Thus, although the redistribution of lipid-like probes by diffusion is not rate limiting in virus-cell fusion, redistribution by diffusion is close to rate limiting in spike-protein mediated cell-cell fusion.