Bennie R. Ware

Actin filament capping and cleaving activity of cytochalasins B, D, E, and H

The concentration dependences of the activities of cytochalasin B, D, E, and H in capping and cleaving actin filaments have been assayed using fluorescence photobleaching recovery. Filament capping was detected by the increase in mobile G-actin. Cytochalasin D (CD) showed the strongest filament capping activity, with an apparent dissociation constant from filament ends of 50 nM. The order of capping activity was CD greater than CH greater than CE much greater than CB. Filament cleavage was detected by the increase in the diffusion coefficients of actin filaments. By this criterion the order of filament cleavage activity was CD, CE greater than CH much greater than CB. Cytochalasin B shows some activity in cleavage of filaments over a concentration range (0-100 microM) at which it shows no appreciable capping activity. This activity, together with results from other groups, is interpreted to mean that CB binds to protomers within the filament, but not to the barbed end. The reversal of activities for CH and CE, combined with the activity profile of CB, constitute the strongest evidence to date that there is more than one cytochalasin binding site on the actin molecule.

pH dependence of actin self-assembly.

Fluorescence enhancement and fluorescence photobleaching recovery have been utilized to examine actin self-assembly over the pH range 6.6-8.0. The kinetics of assembly are faster and the critical concentrations are lower at lower pH. Filament diffusion coefficients are not a function of pH, indicating that average filament lengths are not pH dependent. Although critical actin concentrations are a sensitive function of the concentrations of various cations in the medium, the relative pH dependences of critical concentrations are similar for all combinations of cations employed. The pH dependence of actin self-assembly is sufficiently great that it should be taken into account when comparing data from different reports and when relating in vitro measurements to cytoplasmic mechanisms.

Actin assembly activity of cytochalasins and cytochalasin analogs assayed using fluorescence photobleaching recovery

The effects on actin self-assembly of 9 common cytochalasins and 9 synthetic analogs have been assayed using fluorescence photobleaching recovery (FPR). The specific assembly activities of cytochalasins determined by this assay are (i) reduction of the fraction of actin molecules incorporated into filaments; (ii) increase of the steady-state diffusion coefficients of filaments, from which filaments shortening may be inferred; and (iii) acceleration of the initial rate of assembly. Of the compounds studied, only cytochalasin D shows strong activity of all three types. The range of activities shown by other compounds indicates clearly that these three activity types are distinct and independent. Inspection of the molecular structures of these 18 compounds for correlation of structure and activity reveals that the three different activities depend on distinct structural features. The Mg2+ dependence of filament-shortening activity by certain cytochalasins may be explained by the Mg2+ chelating ability of two suitably positioned oxygen atoms on the convex face of the bicyclic isoindolone system. Inhibition of filament elongation may involve very specific, high-affinity cytochalasin interactions at a binding site on terminal actin molecules, while accelerating activity may occur by weaker, less specific binding interactions of cytochalasins with monomeric actin.

Direct observation of the transition to counterion condensation

Direct observation of the transition to counterion condensation is demonstrated by measurement of the electrophoretic mobility of 6, 6‐ionene, a polyelectrolyte of charge spacing 8.7 A, under conditions of varying dielectric constant. In this way, the reduced charge density parameter ξ can be varied between 0.82 and 1.85. At precisely ξ=1, we observed a drop in the electrophoretic mobility of greater than a factor of 2. Independent viscosity experiments were used to verify that the polymer does not undergo a major conformational transition under these conditions. The reduction of electrophoretic mobility, presumably the result of the reduction of charge density by counterion condensation, is substantially greater in magnitude than current theories of counterion condensation theory would predict. It is suggested that the physical basis for the departure from theory is the fact that near ξ=1, the spacing of condensed counterions predicted by counterion condensation theory is greater than the Debye screening...

Probe diffusion in an aqueous polyelectrolyte solution

Using the technique of fluorescence photobleaching recovery, we have measured the tracer diffusion coefficients of probe particles in aqueous solutions of polystyrene sulfonate at various ionic strengths. Data were fit well by the expression D/D0=exp[−acν’], where D is the probe diffusion coefficient at polymer concentration c, D0 is the probe diffusion coefficient in the absence of polymer, and a and ν’ are familiar scaling parameters. The parameter ν’ ranged from a maximum of 1.0 for the conditions with maximum intersegment interaction (low salt, high‐molecular weight, high‐charge density) to a minimum of about 0.5 for the high‐salt, low‐charge density cases. The parameter a showed little sensitivity to polymer charge density or ionic strength but was a strong function of probe size. No discontinuities in the probe diffusion coefficients or analytical parameters were observed under any solution conditions, even though experimental variations included conditions that are expected to correspond to the tra...

Mobilities of poly-L-lysine molecules in low-salt solutions

We have studied the ordinary–extraordinary phase transition of poly‐L‐lysine solutions using the techniques of quasielastic light scattering (QELS), electrophoretic light scattering (ELS), and fluorescence photobleaching recovery (FPR). Polymer of molecular weight 90 kg/mol was examined in aqueous solutions at a concentration of 1 g polymer/l. The apparent mutual diffusion coefficient, as measured by QELS, was observed to increase sharply by an order of magnitude (from 0.8 to 7×10−7 cm2/s) as the KCl concentration was increased from 2 to 5 mM, in agreement with previous reports by Schurr and co‐workers. On the other hand, the tracer diffusion coefficient (measured by FPR) varies only by about 50% (from 4 to 6×10−7 cm2/s) in a broader trend over the range from 0 KCl to 10 mM KCl. The electrophoretic mobility (measured by ELS) mirrors the change in the tracer diffusion coefficient (from 1.9 to 2.6 μm cm/V s). The ELS and FPR data indicate that poly‐L‐lysine exists primarily as a random coil with little rest...

Determination of electrolyte friction from measurements of the tracer diffusion coefficients, mutual diffusion coefficients, and electrophoretic mobilities of charged spheres

The technique of fluorescence recovery after photobleaching has been used to measure the tracer diffusion coefficient of fluorescein‐labeled charged polystyrene spheres in dilute solutions as a function of solution ionic strength. As the ratio of the particle radius (a) to the Debye–Huckel screening length (κ−1) was varied from 13 to 0.6, a 20% reduction in tracer diffusion coefficient was observed. The mutual diffusion coefficient, measured by quasielastic light scattering, increased as κa was reduced, demonstrating the dominant effect of thermodynamic factors on this parameter. The tracer data have been compared with theoretical predictions of other workers describing the influence of small‐ion interactions with a charge sphere on its translational friction. The theoretical results of Schurr, and Booth, involve an explicit dependence on the electrokinetic charge of the sphere and yield estimates of this parameter (by least squares) which are significantly smaller than those obtained from laser Doppler e...

DNA–divalent metal cation interactions measured by electrophoretic light scattering

We have used the laser Doppler technique of electrophoretic light scattering to study the electrokinetic and gross conformational manifestations of the binding of divalent cations to DNA in low‐salt solutions. By electrokinetic assay, divalent ion affinities to DNA in the 5–500 μM range increase in the order Mn+2<Co+2<Mg+2<Cu+2. The ELS spectra show that Cu+2 denatures double‐stranded DNA in the 50–200 μM concentration range, thereby augmenting the reduction in electrophoretic mobility. Counterion condensation theory correctly predicts the form of the electrophoretic mobility reduction for territorially bound ions such as Mg+2, but the absolute magnitudes of electrophoretic mobility predicted by an equation deduced from condensation theory overestimate our experimental values.

Actin assembly by lithium ions

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.