Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Abstract

Direct measurements of the stiffness (elastic bending resistance) of demembranated sea urchin sperm flagella were made in the presence of MgATP 2-and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of -0 .9 x 10-2 ' N m l, which is -5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP -'does not substitute for MgATP 2 " in producing the relaxed state . A progressive inhibition of movement is observed after addition of MgATP 2-to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP 2-and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP 2-that is not enough to induce spontaneous beating . Vanadate enhances competitive inhibition of beat frequency by MgADP -but not by ADP3-, ATP -, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dyneinnucleotide complexes induced by MgATP 2-and MgADP -. The state accessible by MgATP2-binding must be a state in which the cross-bridges are detached and the flagellum is relaxed . The state accessible by MgADP -binding must be a crossbridged state . Bound vanadate prevents the transition between these two states . Inhibition and relaxation by vanadate in the presence of MgATP2-results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the detached state .The sliding microtubule hypothesis for flagellar and ciliary movement has been firmly established by a series of experimental (42, 49, 52) and theoretical works (4,5,31, 40), since the first important observations by P. Satir (43,44) . This hypothesis predicts that the dynein arm plays a vital role in generating the bending wave of flagella, because the dynein arm is involved in the transduction of the chemical energy provided by ATP dephosphorylation into mechanical energy (15, 16) . The conformational change of the dynein arm is assumed to involve the cyclic change of its orientation, including association and dissociation with the adjacent microtubule, as in the actomyosin system (26). This mechanochemical behavior of the dynein arm is thought to be tightly coupled with the cyclic dephosphorylation of ATP, because the rate of ATP hydrolysis is tightly coupled with flagellar movement and beat frequency (7, 10, 11), and because dynein ATPase not only has a latency (I, 21, 32, 47, 48) but also can be increased in activity by microtubular protein (l, 39) and tubulin (25).The dynein microtubule coupling mediated with ATP has also been suggested by some physiologi-J . CELL . BIOLOGY

Thinking about flagellar oscillation

“…Electron microscopy [Goodenough and Heuser, 1982;Burgess et al, 1991;Burgess, 1995] indicates that the ''stalks'' of the dynein motor heads are attached to substrate sites under most conditions. The OFF mode may be similar to the weakly attached state obtained in the presence of ATP and vanadate, which has a very low shear resistance [Okuno, 1980] and supports longitudinal diffusion of microtubules entrapped by dyneins attached to a surface [Vale et al, 1989]. In this view, the ON mode would incorporate a more strongly attached, force producing state that approaches the ''rigor'' state observed in the absence of ATP.…”

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.

“…This and other reports that AMP-PNP also caused the dissociation of dynein arms from isolated outer-doublet microtubules (26), and that relaxation was not blocked (24) (12, 16), were reasonably accommodated by a model that interpreted flagellar relaxation as corresponding to the first step of the mechanochemical cross-bridge cycle of dynein (24). This mechanochemical interpretation of relaxation appears to have remained prevalent despite recent reports by several groups that AMP-PNP does not effect flagellar relaxation (19,20,22,25) .The relationship of ATP binding to flagellar relaxation is important to investigations of dynein mechanochemistry (18,19,24) as well as to an understanding of the role(s) in motility of putative elastic components in the axoneme, since a simple dynein cross-bridge release mechanism implies that flagellar straightening is driven by forces stored in elastic components, such as the nexin links or radial spokes.We have reinvestigated the roles of ATP binding and hydrolysis in the relaxation of rigor-wave sea urchin sperm flagella by using a novel relaxation assay that has permitted the quantitation of the rate as well as the extent of flagellar straightening. Evidence is presented indicating that flagellar relaxation depends on ATP hydrolysis.…”

“…It was found that (a) the rate of flagellar relaxation induced by 4 x 10 -6 M ATP was inhibited 80% by vanadate concentrations of 3 x 10 -5 M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and E-ATP), were also capable of effecting relaxation ; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein arm in the course of flagellar relaxation ; and (d) the ratio of the rate of relaxation to the rate of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP concentration was raised from 2 x 10 -6 to 9 x 10 -6 M. It is concluded that (a) flagellar relaxation depends on ATP hydrolysis ; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein ; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon . A general model of relaxation is proposed based on active microtubule sliding.The relaxation (straightening) of rigor-wave sea urchin sperm flagella is currently favored as a model system for investigating the mechanochemical cycle of flagellar motility (18,19,23,24). This is due to the view that relaxation results from the release of dynein cross-bridges in the flagellar axoneme in response to ATP binding .…”

“…The relaxation (straightening) of rigor-wave sea urchin sperm flagella is currently favored as a model system for investigating the mechanochemical cycle of flagellar motility (18,19,23,24). This is due to the view that relaxation results from the release of dynein cross-bridges in the flagellar axoneme in response to ATP binding .…”

“…The relationship of ATP binding to flagellar relaxation is important to investigations of dynein mechanochemistry (18,19,24) as well as to an understanding of the role(s) in motility of putative elastic components in the axoneme, since a simple dynein cross-bridge release mechanism implies that flagellar straightening is driven by forces stored in elastic components, such as the nexin links or radial spokes.…”