We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.

Abstract. Two types of Chlamydomonas reinhardtii tlagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, 12, and 13, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. E Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and 12, respectively. The 13 idaA isolates comprised three genetically different groups (idal, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged crosssection electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In idal-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant idal-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both idal-3 and ida4 swam at reduced speed.Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants idal-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.

Little is known about the electrostatic/dynamic properties of microtubules, which are considered to underlie their electrostatic interactions with various proteins such as motor proteins, microtubule-associated proteins, and microtubules themselves (lateral association of microtubules). To measure the dielectric properties of microtubules, we developed an experiment system in which the electroorientation of microtubules was observed under a dark-field microscope. Upon application of an alternating electric field (0.5-1.9 x 10(5) V/m, 10 kHz-3 MHz), the microtubules were oriented parallel to the field line in a few seconds because of the dipole moment induced along their long axes. The process of this orientation was analyzed based on a dielectric ellipsoid model, and the conductivity and dielectric constant of each microtubule were calculated. The analyses revealed that the microtubules were highly conductive, which is consistent with the counterion polarization model-counterions bound to highly negatively charged microtubules can move along the long axis, and this mobility might be the origin of the high conductivity. Our experiment system provides a useful tool to quantitatively evaluate the polyelectrolyte nature of microtubules, thus paving the way for future studies aiming to understand the physicochemical mechanism underlying the electrostatic interactions of microtubules with various proteins.

Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesinmicrotubule rigor complex using the optical trap, and ATP was released by f lash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 M ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 M ؊1 ⅐s ؊1 , followed by force generation at 45 s ؊1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.Motor proteins, such as kinesin, myosin, dynein, and RNA polymerase, convert the chemical energy of ATP hydrolysis into mechanical work. To investigate the mechanism of energy transduction of motor proteins, the elementary mechanical steps like force generation and sliding should be related to chemical reactions such as ATP binding, and P i and ADP release. The biochemistry of ATP hydrolysis by kinesin in solution has been investigated using native and recombinant proteins, with or without microtubules. The ATPase rate of kinesin is very low (Ͻ0.01 s Ϫ1 ) in the absence of microtubules at room temperature and is activated more than 2,000-fold by microtubules to Ͼ20 s Ϫ1 (1-5). The intermediate states of the kinesin-microtubule complex are similar to those of actomyosin, that is, M⅐K, M⅐K⅐ATP, M⅐K⅐ADP⅐P i , and M⅐K⅐ADP, where M, K, and P i are microtubule, kinesin, and inorganic phosphate, respectively. The rate-limiting step of the ATPase cycle is ADP release in the absence of microtubules, and P i or ADP release in the presence of microtubules (1-5).These biochemical results cannot be correlated directly to the mechanical cycle of kinesin, because the movements are not detected in solution. A powerful method for the investigation of mechanochemical coupling is to detect the mechanical change after laser photolysis of caged compounds, e.g., caged ATP, caged ADP, and caged P i (6, 7). Caged compounds photolyze within several milliseconds after a pulse of ultraviolet light. The mechanical reactions of actomyosin in muscle have been detected after release of ATP by photolysis of caged ATP. The rate of actomyosin dissociation after ATP release and rate of force generation were determined (6).In the experimental systems containing many molecules, individual events are averaged and the state of each molecule is not determined. Analysis of the events produced by single molecules reveals the molecular functions more directly. The kinetics of single ion channels were elucidated by stochastic analys...