Sergey V. Mikhailenko

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions.

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch.

The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADP-activation (1 mM MgATP and 2 mM MgADP in the absence of Ca2+) and also at Ca2+-activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either “resisting” or “yielding,” which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere “yielding” occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-α-actinin antibody significantly suppressed the clustered sarcomere “yielding.” These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca(2+) and incubated overnight at 4 degrees C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca(2+)-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.

D-loop of actin differently regulates the motor function of myosins II and V

To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V “walked” with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP·Pi state of both myosins.

Modulation of the mechano-chemical properties of myosin V by drebrin-E

The regulation of actin filament networks by various proteins has essential roles in the growth cone dynamics. In this study we focused on the actin-myosin interaction which has been suggested to be an important player in the neurite extension. We examined in vitro how the decoration of actin filaments with a side-binding protein, drebrin-E, affects the motile properties of an intracellular transporter myosin V. Single myosin V molecules landed on the drebrin-E-decorated actin filaments with a lower frequency and ran over shorter distances; however, their velocities were normal. Furthermore, the analysis of the movement of myosin V molecules in the optical trap revealed that the decoration of actin filaments with drebrin-E markedly increased the load-sensitivity of the myosin V stepping. These results are attributable to the delay in the attachment of the motors leading head (ADP·P(i) state) to actin, induced by the competitive binding of drebrin-E to actin, whereas the rate of ADP release from the trailing head (the rate-limiting step in the ATPase cycle of myosin V) is unaffected. Our study indicates that, in addition to the regulation of binding affinity of myosin V, drebrin-E also modulates the chemo-mechanical coupling in the motile myosin V molecules, presumably affecting the movement of the growth cone.

The Force Production by the Depolymerization Activity of MCAK

During cell division the replicating chromosomes must be precisely arranged and separated polewards. Though many cellular processes involving motility require force generation by motor proteins, the chromosome movement is suggested to use the energy stored at plus ends of the microtubules to which kinetochores are attached. This energy is converted into the chromosome movement via passive couplers, whereas the role of the microtubule-based motors is supposed to be limited to the regulation of microtubule dynamics. The microtubule-depolymerizing kinesins, such as mitotic centromere-associated kinesin (MCAK), which is a founding member of kinesin-13 family, facilitate microtubule dynamics in a spindle and are required for the chromosome congression and segregation; however, the key question - whether the depolymerizing activity generates tension to pull the chromosomes - has remained unsolved. To probe the link between the generation of tension and the microtubule-disassembling activity of MCAK, we developed a single-molecule assay to examine the interaction between a single microtubule and multiple MCAK molecules under the fluorescence microscope equipped with the dual-trap optical tweezers. Here we show that the microtubule-disassembling activity of MCAK generates significant tension. The depolymerization force increases with the number of interacting MCAK molecules. These results provide a simple and attractive model for the generation of the driving force and the regulation of chromosome segregation in a spindle by the activity of MCAK.