Defocused orientation and position imaging (DOPI) of myosin V

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'wiggly'' walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm.FIONA ͉ fluorescence ͉ motility ͉ single molecule assay ͉ unconventional myosin D espite fairly intensive study, how myosin VI moves remains elusive. It is known that myosin VI moves toward the minus end of actin in contrast to all other myosins (1). Myosin VI also has a large and variable step size of Ϸ30 Ϯ 12 nm with occasional back steps of approximately 13 Ϯ 8 nm (2, 3), which is made possible by a combination of a short calmodulin-containing lever arm (4) and a lever-arm extension that is created by the unfolding of a threehelix bundle (5). The processive stepping is a hand-over-hand motion (6, 7) and has recently been reported to involve a ''wiggly'' movement around actin (8). This is in contrast to myosin V, arguably the best understood myosin motor (9), which takes a 36-nm step size (10), in a hand-over-hand fashion (11), tilting its large lever arm approximately 70°(12, 13), in a relatively straight fashion, with some twisting about the actin axis (13-15).The atomic structure of myosin VI has been revealed in the post-power stroke states (16) and a truncated form in the pre-power stroke states (17). It is largely the same as myosin V, but myosin VI has two inserts, one near the nucleotide pocket, which allows it to fine-tune its response to ADP and ATP, and a second unique insert at the end of the converter domain, where the lever arm connects to the motor domain, which repositions the lever arm and reverses the power stroke (18,19).While it is unclear as to the exact degree of rotation that the power stroke undergoes, it is undoubtedly large. Ménétrey et al., based on the crystal structures, suggested it was nearly 180°(16, 17). Bryant et al., based on a series of single-headed constructs in an optical trap, also concluded that the lever arm undergoes a 180°r edirection during the power stroke (20). Sun et al., based on single-molecule fluorescence angular changes, stated that their data were most consistent with variable lever arm positions, with the majority involving an approximate 180°reorientation of the lever arm (8). Furthermore, they observed ''chaotic left-right wiggling'' of the lever arm as the myosin VI moves along the actin filament.The aspect of the myosin VI processive mechanism that had b...

contrasting

confidence: 57%

Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 99%

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Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm

Reifenberger

Toprak

Kim

et al. 2009

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“…Approaches have been developed that rely on excitation and/or emission with multiple polarizations (19,20), introduction of defocus and pattern matching (21), direct imaging of pupil functions (22), and use of annular illumination to create characteristic field distributions (23) to name a few. The alternating measurement of 2D position and orientation has also been addressed (24). Two groups considered simultaneous 2D localization and orientation fitting for molecules located in the focal plane (25) or molecules at a known defocus (26).…”

mentioning

confidence: 99%

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Backlund¹,

Lew²,

Backer

et al. 2012

190

214

Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photonlimited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

“…The precisely metered stepping action is a function of the length of the neck (5), an extended regulatory domain of six calmodulin-binding IQ motifs that link each head to the coiled-coil tail. The favored Ϸ36-nm step allows the motor to pick out a linear pathway along a helical actin filament; however, recent work has revealed that off-axis binding can and does occur (6,7). Like other molecular motors, myosin-V works directionally, with forward steps being favored over backward steps.…”

mentioning

confidence: 99%

Myosin's mechanical ratchet

Cross

2006

M yosin molecular motors bind to and exert force upon actin filaments. Between 24 (1) and 37 (2) myosin subfamilies are currently recognized (3), the myriad members of which are specialized for tasks ranging from muscle contraction through to mechano-sensation and signaling, cell polarization, the sliding and tensioning of cytoskeletal elements, organelle transport, exocytosis and endocytosis, and vesicle transport. The molecular mechanism by which myosins step along actin filaments is accordingly a central problem, studies of which have been greatly facilitated in recent years by the discovery of processive myosins. Processive myosins can take many sequential steps along an actin filament, remaining attached throughout. One such is myosin-V (4), a twin-headed myosin that walks along actin filaments toward their fast-growing (blunt) ends, measuring out paces that correspond more or less exactly to the Ϸ36-nm helical repeat of the actin filament. The precisely metered stepping action is a function of the length of the neck (5), an extended regulatory domain of six calmodulin-binding IQ motifs that link each head to the coiled-coil tail. The favored Ϸ36-nm step allows the motor to pick out a linear pathway along a helical actin filament; however, recent work has revealed that off-axis binding can and does occur (6, 7). Like other molecular motors, myosin-V works directionally, with forward steps being favored over backward steps. Sustained directional stepping by any motor against a load requires an energy source, which, in the case of the myosins, is supplied by the hydrolytic conversion of ATP into ADP and P i . Coupling the mechanical stepping cycle to ATP turnover in this way allows the myosin-V motor to step continuously against a load of up to Ϸ2-3 pN, consistent with its using one ATP per 36-nm step. Above Ϸ2-3 pN, the motor stalls, pausing with both heads attached to the actin filament (8 -10) but unable to step forward because the work involved exceeds the energy available from ATP hydrolysis. Importantly, the evidence suggests that, in this stalled state, ATP turnover is halted, so that the myosin-V motor only consumes ATP when it is actively stepping. This invariant mapping of exactly one ATP molecule per step, and one step per ATP molecule, is called tight coupling. An obvious question is, what happens if one pulls backward on a walking myosin-V molecule with greater force than the stall force? Will the motor simply be torn from its track, or will it step back? And if it steps back, will it step in a controlled way, and will the tight requirement for one ATP molecule per step be maintained? In a recent issue of PNAS, Gebhardt et al.(11) reported answers to exactly these questions. Remarkably, they found that pulling backward on a walking myosin-V molecule causes the motor to reverse its mechanical action, again taking measured-out 36-nm steps, but without a requirement for ATP binding. Backstepping continues, at a rate that depends on the load, until the load diminishes to the point where the motor can...