Myosin-V makes two brownian 90° rotations per 36-nm step

Komori, Yasunori; Iwane, Atsuko H.; Yanagida, Toshio

doi:10.1038/nsmb1298

Cited by 30 publications

(23 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This flexibility in the rotation angle may be functionally advantageous, as it should help myosin avoid any obstructions in the crowded actin network of a cell by allowing it to adopt to different conformations to maximize the probability that the heads will alternate between the lead and rear positions during motility. Our model can also explain why in our previous study (21) an actin filament underwent two 90°Brownian rotations per 36 nm myosin V step, while in the present study only one 90°rotation was seen. In the previous study, myosin V was fixed onto a glass surface at its tail end, whereas in this one the tail end is free.…”

Section: Discussionsupporting

confidence: 50%

“…We previously found that a fluorescently labeled actin filament can be rotated by myosin V fixed to a glass surface (21). In the present study, we directly observed myosin rotations by simultaneously measuring myosin steps and orientations, finding myosin V rotates 90°for each 36 nm step.…”

Section: Discussionmentioning

confidence: 65%

“…This difference would cause the heads to be antiparallel in ref. 21, but perpendicular here when myosin V is in the one-head bound state.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Ohmachi

Komori

Iwane

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10°accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90°for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.quantum rods | single molecule imaging S ingle molecule fluorescence imaging techniques are being increasingly used to observe the dynamic properties of single molecules like spatial orientation, which provides information on a protein's three-dimensional (3D) motility and its conformational changes (1, 2). These techniques can be grouped into two different classes: intensity distribution techniques and interference pattern techniques. In the first class, 3D orientation is determined by comparing fluorescence intensities among several excitation or detection polarizations. For example, in single-molecule fluorescence polarization microscopy (3), the dye is excited by multiple polarized beams incident from different directions. The resulting emission is split with respect to its polarization and detected with avalanche photodiodes, which allows for sensitivity to the 3D orientation of a single dye's transition dipole moments. The second class takes advantage of single molecule emission patterns created by defocusing. Defocused imaging reveals additional structures in single-molecule emission patterns that depend on the orientation of the emitting dipole (4-7). The 3D orientation is obtained by comparing the defocused images with corresponding calculated model images. This technique can be used to determine both the position and orientation of the dye. However, because the defocused image is spread over a greater number of pixels, the image inherently has poor position accuracy. Furthermore, in most orientation-determination techniques, time consuming fitting...

show abstract

Section: Discussionsupporting

confidence: 50%

Section: Discussionmentioning

confidence: 65%

See 1 more Smart Citation

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Ohmachi

Komori

Iwane

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The behavior of kinesin is in sharp contrast with a distantly related processive motor that is also known to move hand-overhand, myosin-V, which has recently been reported to undergo random, Ϯ90°stalk rotations per step (32), indicating that the heads indiscriminately take left and right steps. The source of these rotations was attributed to accumulation and relief of torsional strain within the otherwise rigid dimer.…”

Section: Discussionmentioning

confidence: 77%

Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping

Gutiérrez–Medina¹,

Fehr

Block

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are Ϸ30-fold stiffer than its flexible ''hinge'' regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.microtubule ͉ molecular motor ͉ rotation ͉ single molecule ͉ yaw with one degree accuracy C onventional kinesin (kinesin-1) is a motor protein that transports cargo along microtubules (MTs) in cells, converting the chemical energy of nucleotide hydrolysis into mechanical work (1). The structure of kinesin consists of twin catalytic motor domains (heads) that bind to the MT substrate with nucleotidedependent affinity, joined by neck linkers (NLs) to the N terminus of an extended, ␣-helical coiled-coil (CC) stalk (Fig. 1A). Considerable recent progress has been made in understanding the mechanochemistry of the kinesin motor domains, which enable individual molecules to move processively, undertaking hundreds of successive 8-nm steps while hydrolyzing one ATP molecule per step (2-4). However, little is known about the structure of the stalk or its role in the currently favored model for kinesin movement, the so-called ''asymmetric hand-overhand'' stepping mechanism (5-7). In this mechanism, the heads of kinesin advance in strict alternation, exchanging leading, and trailing roles at each step, analogous to a person walking (1). An asymmetric mechanism requires that the ''left'' and ''right'' steps be slightly different; however, in contrast with a symmetric hand-over-hand walk, it does not require continuous rotation of the kinesin stalk. Consistent with this model, a landmark experiment by the Gelles group (8) that tracked the angular motions of MTs driven by single kinesin motors affixed to a surface did not find evidence for large-scale rotations.Although no net rotations of the kinesin stalk are expected during asymmetric hand-over-hand motion, significant momentary angular changes are nevertheless possible. If kinesin were a nearly rigid molecule, Ϯ180°reorientations of the stalk would be anticipated whenever the two heads exchanged positions during stepping (1, 9). It has been speculated that flexible elements within the kinesin structure may relieve any such momentary torsional strain, facilitating forward motion. Although previous single-molecule studies foun...

show abstract

“…Recently, the diffusive motion of unbound myosin V heads has been directly observed during the stepping process ( Dunn & Spudich 2007;Shiroguchi & Kinosita 2007). The random twisting motion of the neck domain around its axis was observed associated with the step movement (Komori et al 2007).…”

Section: Processive Movement Of Unconventional Myosinsmentioning

confidence: 99%

Single molecule measurements and molecular motors

Yanagida

Iwaki

Ishii

2008

Phil. Trans. R. Soc. B

Self Cite

View full text Add to dashboard Cite

Single molecule imaging and manipulation are powerful tools in describing the operations of molecular machines like molecular motors. The single molecule measurements allow a dynamic behaviour of individual biomolecules to be measured. In this paper, we describe how we have developed single molecule measurements to understand the mechanism of molecular motors. The step movement of molecular motors associated with a single cycle of ATP hydrolysis has been identified. The single molecule measurements that have sensitivity to monitor thermal fluctuation have revealed that thermal Brownian motion is involved in the step movement of molecular motors. Several mechanisms have been suggested in different motors to bias random thermal motion to directional movement.

show abstract

Myosin-V makes two brownian 90° rotations per 36-nm step

Cited by 30 publications

References 34 publications

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping

Single molecule measurements and molecular motors

Contact Info

Product

Resources

About