Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

Compartmentalized signaling involving cholesterol-rich, liquid-ordered membrane domains occurs during cell activation triggered by receptor cross-linking, growth factors, or other extracellular stimuli (1-3). The redistribution of similar liquidordered domains, called lipid ''rafts,'' accompanies and is required for cell polarization and directed migration (4 -8). Although we do not know the precise molecular mechanisms by which the redistributions of plasma membrane domains occur, an active role of the actin-based membrane skeleton has long been postulated (reviewed in Ref. 9).Redistributions of activated or cross-linked receptors are accompanied by corresponding changes in the localizations of actin, nonmuscle myosin II, spectrin, and associated cytoskeletal proteins (9). Furthermore, disruption of actin filament integrity inhibits many lipid raft-mediated processes, including epidermal growth factor receptor capping in A431 cells (10), insulin receptor capping in lymphocytes (11), activation of fibroblasts (12), polarization of T lymphocytes (5), and downregulation of Fc⑀RI-mediated signaling in mast cells (13). A requirement for myosin II is shown by the greatly diminished receptor redistributions and/or developments of cell polarity that have been observed in cells that either lack myosin II (14, 15) or express a dominant-negative mutant of myosin II function (16 -18). Erythrocyte spectrin (19) and the nonerythroid spectrin called fodrin 1 (20, 21) also have been implicated in lipid raft-mediated processes. Taken together, these observations suggest that actin filaments, perhaps as components of a spectrin-based membrane skeleton, are required for the

The role of the highly conserved residues in the ␥-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser 181 , Lys 185 , Asn 235 , Ser 236 , and Arg 238 ) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial P i burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg 2؉ -ATPase activity, initial P i burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg 2؉ -ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actintranslocating activity. These results indicate that 1) the hydroxyl group of Ser 236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg 238, which interacts with Glu 459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu 459 and Arg 238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.Myosins are molecular motors that translocate actin filaments using energy from ATP hydrolysis and participate in diverse biological contractile events, such as muscle contraction, cytokinesis, cell locomotion, and organelle movements. During the last few years, a number of myosin-like proteins have been identified, and it is now clear that myosin consists of a large family of actin-based motors (1-4). While the C-terminal tail portion of these myosins is quite diverse, the N-terminal 80-kDa motor domain, which contains the ATP binding site and actin binding sites, is highly conserved among various myosins (4). Therefore, it is thought that the mechanism by which the chemical energy of ATP is converted to mechanical energy is universal in all myosin family members. Myosinbased motility occurs as the result of cyclic association and dissociation of myosin molecules and F-actin molecules with concomitant hydrolysis of ATP. This cycle can lead to relative sliding of the actin and myosin and the production of work. Equation 1 contains the generally accepted mechanism of actomyosin ATP hydrolysis, where AM and M represent actomysin and myosin, respectively. ATP binding to actomyosin is rapid and produces a rapid dissociation of myosin from actin (Ͼ1000 s Ϫ1 ). The rate of M-ATP hydrolysis to M-ADP-P i is also much faster than the steady state rate of hydrolysis by myosin, which in the absence of actin, is limited by slow release of inorganic pho...

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated ϳ10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K actin . These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K actin was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K actin , whereas the V max was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P i release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.Class III myosin was originally found in Drosophila photoreceptor cells (1), and a majority of the cell biological work related to class III myosin has been done with the Drosophila system. Class III myosin was subsequently identified from humans (2, 3), striped bass (4), and Limulus (5). In vertebrate, two isoforms of class III myosin, myosin IIIA and myosin IIIB, have been cloned (2, 3). Among them, most studies have been done with myosin IIIA. Both isoforms are highly expressed in the retina. Myosin IIIA is also expressed in inner ear hair cells, and it is responsible for progressive nonsyndromic hearing loss in humans (6). Immunohistochemical studies revealed that myosin IIIA is concentrated in the distal ends of rod and cone ellipsoid and colocalizes with the plus-distal ends of inner segment actin filament bundles, where actin forms the microvilli-like calycal processes (4). Interestingly, the transfection of green fluorescent protein-myosin IIIA into HeLa cells revealed that myosin IIIA localizes at the tip of filopodia (7), suggesting that myosin IIIA accumulates at the plus end of actin bundles. The major cytoskeletal structure of filopodia is the actin bundles, and the plus ends of the actin filaments are localized at the tip; therefore, the localization of myosin IIIA at the tip of filopodia suggests that this myosin traveled on actin filaments and accumulated at the end of the actin track. This is consistent with our result that my...

The motor function of smooth muscle myosin is activated by phosphorylation of the regulatory light chain (RLC) at Ser 19 . However, the molecular mechanism by which the phosphorylation activates the motor function is not yet understood. In the present study, we focused our attention on the role of the central helix of RLC for regulation. The flexible region at the middle of the central helix (Gly 95 -Pro 98 ) was substituted or deleted to various extents, and the effects of the deletion or substitution on the regulation of the motor activity of myosin were examined. Deletion of Gly 95 -Asp 97 , Gly 95 -Thr 96 , or Thr 96 -Asp 97 decreased the actin-translocating activity of myosin a little, but the phosphorylation-dependent regulation of the motor activity was not disrupted. In contrast, the deletion of Gly 95 -Pro 98 of RLC completely abolished the actin translocating activity of phosphorylated myosin. However, the unregulated myosin long subfragment 1 containing this RLC mutant showed motor activity the same as that containing the wild type RLC. Since long subfragment 1 motor activity is unregulated by phosphorylation, i.e. constitutively active, these results suggest that the deletion of these residues at the central helix of RLC disrupts the phosphorylation-mediated activation mechanism but not the motor function of myosin itself. On the other hand, the elimination of Pro 98 or substitution of Gly 95 -Pro 98 by Ala resulted in the activation of actin translocating activity of dephosphorylated myosin, whereas it did not affect the motor activity of phosphorylated myosin. Together, these results clearly indicate the importance of the hinge at the central helix of RLC on the phosphorylation-mediated regulation of smooth muscle myosin.The motor activity of vertebrate smooth muscle as well as non-muscle cell myosin is regulated by phosphorylation of the 20,000 dalton light chain (LC20) of myosin (1-5). While the phosphorylation of myosin at LC20 occurs at several sites which are catalyzed by various protein kinases, the activation effect is rather specific to the site of phosphorylation, and only the phosphorylation at serine 19 and/or threonine 18 can activate the motor activity of myosin (6 -8). Recent three-dimensional structural analysis of the skeletal muscle myosin head domain greatly facilitated the understanding of the structurefunction relationship of the myosin motor (9). It was revealed that the ATPase active site and actin binding site, two functionally essential regions of the myosin motor, exist toward the top of the myosin head while the regulatory light chain (i.e. LC20) associates with the myosin heavy chain at the lower end of myosin head, i.e. adjacent to the head-rod junction. This structural feature indicates that it is unlikely that the regulation is achieved by the direct interaction between the motor effector sites and the phosphorylation site. This leads to the hypothesis that the change in the conformation of LC20 induced by phosphorylation is transmitted to the motor effector sites via ...