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A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1-100 nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10 microliters with temporal resolution >1 Hz. For 2-100 microM f-actin gels a final viscosity of 1.3-9.4 mPa s and a final elastic modulus of 2.229-2.254 GPa (corresponding to 1493-1501 m/s sound velocity) have been determined. For 10- to 100-microM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5-124 mPa s and a final elastic modulus of 2.288-2. 547 GPa (approximately 1513-1596 m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3 m/s (approximately 1.69 kPa) and decreased up to -7 m/s (approximately 49 kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to -12 m/s approximately 2.3-0.1 MPa, for 10- to 100-microM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2 mM ATP (when compared to 0.2 mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17 microM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26 GPa) was less than for f-actin polymerized in presence of 0.2 mM ATP (2.28 GPa).

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.

In density-inhibited 3T3 fibroblast cultures F-actin is localized almost exclusively at the cell-to-cell borders. Depolymerization of microtubules results in a rearrangement of the F-actin pattern. Numerous actin bundles are formed, most of them are anchored to the cell borders. This rearrangement is preceded by a transient rise in F-actin content.

Cyclic rearrangements of the microtubules (Mts) occur in the hyposquamal scleroblasts which synthesize the highly ordered three-dimensional collagen network forming the basal plate of the fish scale. The distribution of Mts containing tyrosinated and detyrosinated alpha-tubulin (Tyr-Mts and Glu-Mts, respectively) was analyzed in relation to the frequency of Mt reorganization in the teleostean elasmoid scale during collagen deposition using two specific monoclonal antibodies. In the very flat hyposquamal scleroblasts of fully developed scales (with a very low synthetic activity) two microtubule populations were identified. Most contain Tyr-tubulin while Glu-tubulin is found in some "stable" Mts. In the tall prismatic scleroblasts of regenerating scales (active in collagen synthesis) only Tyr-Mts have been revealed. In late stage of regeneration, when the synthetic activity decreases and the scleroblasts flatten, an increasing centriole and Mt labeling with Glu-tubulin antibody was observed. The intimate relationship of intracellular microtubule arrangement and extracellular collagen fibril pattern reported earlier (Zylberberg et al., Cell Tissue Res. 253, 597-603 (1988], together with the results presented here, support the hypothesis that a changing Mt pattern is involved in the generation of the collagen plywood.

Endothelial cells derived from Xenopus tadpole hearts (XTH-2: Schlage et al. 1981) have been permeabilized for calcium using the ionophore A 23187. A Ca2+-concentration of 1 microM causes a graded disassembly of microtubules (mts): with increasing periods of incubation mts disintegrate and then depolymerize completely. The frequency of the different stages of depolymerization depends on the length of the Ca2+ incubation period, even after 60 min incubation time some mts remain. The stage of depolymerization of mts in a cell is correlated with its cell cycle phase showing the highest stability during S phase.

A pressure device has been used to obtain information about the forces involved in the maintenance of the aggregated state of melanophores of the angelfish, Pterophyllum scalare. Single aggregated melanophores of isolated scales were submitted to mechanical compression with forces ranging from 50-320 mup. As a function of the gradually increasing force melanophores disperse their pigment, the degree of dispersion being proportional to the intensity of the force. When microtubules are destroyed by treatment with 0.3 mM vinblastine in KCl solution, pigment dispersion in response to the external force is similar to that observed in KCl alone. After changing the medium to NaCl solution, melanin granules remain concentrated in the cell center; the force-induced melanosome dispersion, however, is significantly enhanced. Distinctly lower forces are required to produce an expansion equivalent to that observed in KCl solution. When the medium is changed to vinblastine-KCl again, the dispersion in response to the external force resembles that obtained before NaCl treatment. Removal of Ca++ and Mg++ ions by treatment with 2 mM EDTA or EGTA in Ringer's solution containing 0.1 mM adrenalin produces a remarkable enhancement of melanosome dispersion in response to increasing external force. This effect of EDTA or EGTA is completely reversible. When the medium is changed to normal Ca++-Ringer's, the force/dispersion curve resembles that obtained before EDTA or EGTA treatment. It is concluded that a state of equilibrium exists between the external force and an opposing force produced by the melanophore. The differences in the opposing force under different experimental conditions may be due to a "contractile component". This component seems to be independent of microtubules, as indicated by vinblastine experiments. It "contracts" under aggregating stimulus and "relaxes" under dispersing stimulus. From the data presented in this paper, the order of magnitude of the pressure developed by the contractile component in the completely aggregated state was calculated as between 5-7 p/cm2 in the relaxed state and about 20 p/cm2 during contraction. These values are comparable to those observed in other nonmuscular cells.

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.