Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.

Stimulation of circus movement by activin, bFGF and TGF-beta 2 in isolated animal cap cells of Xenopus laevis.

Lobopodium is a hyaline cytoplasmic protrusion which rotates circumferencially around a cell. This movement is called circus movement, which is seen in dissociated cells of amphibian embryos. Relative abundance of the lobopodia-forming cells changes temporally and spatially within Xenopus embryos, reflecting stage-dependent difference of morphogenetic movements. The lobopodia-forming activity of dissociated animal cap cells was stimulated strongly by activin and bFGF, and weakly by TGF-beta 2. In addition, activin A was found to stimulate cellular attachment to the substratum when the cultivation lasted long. Thus, mesoderm-inducing growth factors stimulate lobopodia formation and cellular movements which may be necessary for gastrulation and neurulation in Xenopus early embryos.

Strikingly different propulsive forces generated by different dynein-deficient mutants in viscous media.

The propulsive force generated by Chlamydomonas mutants deficient in flagellar dynein was estimated from their swimming velocities in viscous media. The force produced by wild-type cells increased by 30-40% when viscosity was raised from 0.9 to 2 cP but decreased as viscosity was further raised above 6 cP. The biphasic dependence of force generation on viscosity was also observed in the mutant ida1, which lacks the I1 component of the inner-arm dynein. The mutant ida4, which lacks the inner-arm I2 component, was extremely susceptible to viscosity and stopped swimming at 6 cP, at which other mutants could swim. In contrast, oda1, which lacks the entire dynein outer arm, produced a fairly constant force of about one-third of the wild-type value, over a viscosity range of 0.9-11 cP. In demembranated and reactivated cell models of the wild type, the propulsive force decreased monotonically as viscosity increased. Thus the increase in force generation at about 2 cP observed in live cells may be caused by some unknown mechanism that is lost in cell models. The cell models of oda1, in contrast, did not show a marked change in force generation with the change in viscosity. These results indicate that the force generation by the outer-arm dynein greatly depends on viscosity or the velocity of movement, whereas the complete set of inner-arm dynein present in the oda1 axoneme produces a fairly constant force at different viscosities. These different properties of inner and outer dynein arms should be important in the mechanism that produces flagellar beating.