Long single α-helical tail domains bridge the gap between structure and function of myosin VI

Spink, Benjamin J.; Sivaramakrishnan, Sivaraj; Lipfert, Jan; Doniach, Sebastian; Spudich, James A.

doi:10.1038/nsmb.1429

Cited by 115 publications

(205 citation statements)

References 54 publications

(84 reference statements)

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“…In order to account for the observed beta angles of the CaMcontaining lever arms, we indicate a second point of dimerization immediately following the lever arm extension (red), as we have recently demonstrated (7). recently proposed by Spink et al (25). In this model, the CaMcontaining lever arm of myosin VI is extended by long single alpha helices and dimerization between the myosin VI monomers occurs only in the region of their cargo binding domains (approximately the position of the GCN4 leucine zipper in our HMM construct).…”

Section: Resultsmentioning

confidence: 99%

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

Reifenberger

Toprak

Kim

et al. 2009

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

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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...

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Section: Resultsmentioning

confidence: 99%

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

Reifenberger

Toprak

Kim

et al. 2009

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

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“…Their logic was that a construct containing 835-1,035 did not dimerize and that the region of myosin VI that was originally thought to form a coiled coil has been proposed to be a stable single ␣-helix (SAH) (18,23,24). Thus, Spink et al (18) postulated that the SAH domain forms an extension of the short myosin VI lever arm, enabling the dimer to take large steps on actin. However, we subsequently demonstrated that a lever arm extension is formed upon dimerization by unfolding of a three-helix bundle (6), formerly known as the proximal tail (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Spink et al (18) concluded that dimerization of the full-length molecule involves only dimerization of the cargo-binding domains. However the construct that dimerized in their study contained residues 835-1,285, and the cargo-binding domain is from Ϸ1,035 to 1,285 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1, using the terminology of Spink et al (18). We have demonstrated that internal dimerization (probably coiled coil) occurs between residues 913 and 936 (6).…”

mentioning

confidence: 99%

“…Subsequently, it was shown that a headless myosin VI construct containing the entire tail and cargo-binding domains dimerized with relatively high affinity (18). Thus there may be two separate regions of the myosin VI molecule that can contribute to dimerization.…”

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confidence: 99%

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Cargo binding induces dimerization of myosin VI

Phichith

Travaglia²,

Yang³

et al. 2009

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

105

104

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Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.Dab2 ͉ directionality ͉ motility ͉ optineurin ͉ unconventional myosin W ithin the myosin superfamily there are at least 35 classes of molecular motors that move along actin filaments (1). Myosin VI is the only class of myosin known to move toward the minus-end of actin filaments (2, 3). Myosin VI dimers take large and variable steps on actin (average of 30-36 nm) using a short lever arm and a unique lever arm extension (4-6). Not only is the myosin VI dimer capable of processive movement (i.e., can move as a single molecule) along an actin filament (4-6), it also functions as a load-dependent actin anchoring protein (7). Thus myosin VI can potentially fulfill a number of specialized cell biological functions (8-10). Paradoxically, although these functional features suggest that myosin VI is designed to work in cells as a dimer, myosin VI as isolated from cells is a monomer, and expressed full-length myosin VI is also monomeric (4,5,11).A number of cargo adaptor proteins that recruit myosin VI have been identified (8). For example, it has been demonstrated that optineurin is essential for myosin VI localization to the Golgi complex (12), and binds to a site within the globular tail of myosin VI. Dab2 (13,14) and Sap97 (15) mediate the recruitment of myosin VI to clathrin-coated pits and vesicles, whereas GipC serves this role on uncoated vesicles (16,17).The myosin VI molecule has discrete structural domains, as diagrammed in Fig. 1, using the terminology of Spink et al. (18). We have demonstrated that internal dimerization (probably coiled coil) occurs between residues 913 and 936 (6). However, we noted that this dimerization is weak and forms only if the molecules are held in close proximity (5, 6). We postulated that in vivo dimerization is initiated upon binding of myosin VI to a dimeric cargo, which would then trigger the weak internal dimerization (5, 6).Subsequently, it was shown that a headless myosin VI construct containing the entire tail and cargo-binding domains dimerized with relatively high affinity (18). Thus there may be two separate regions of the myosin VI molecule...

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Plus‐end directed myosins accelerate actin filament sliding by single‐headed myosin VI

et al. 2012

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Myosin VI (Myo6) is unique among myosins in that it moves toward the minus (pointed) end of the actin filament. Thus to exert tension on, or move cargo along an actin filament, Myo6 is working against potentially multiple plus (barbed)-end myosins. To test the effect of plus-end motors on Myo6, the gliding actin filament assay was used to assess the motility of single-headed Myo6 in the absence and presence of cardiac myosin II (Myo2) and myosin Va (Myo5a). Myo6 alone exhibited a filament gliding velocities of 60.34 +/− 13.68 nm/s. Addition of either Myo2 or Myo5a, at densities below that required to promote plus-end movement resulted in an increase in Myo6 velocity (~100-150% increase). Movement in the presence of these plus-end myosins was minus-end directed as determined using polarity tagged filaments. High densities of Myo2 or Myo5a were required to convert to plus-end directed motility indicating that Myo6 is a potent inhibitor of Myo2 and Myo5a. Previous studies have shown that two-headed Myo6 slows and then stalls in an anchored state under load. Consistent with these studies, velocity of a two headed heavy mero myosin form of Myo6 was unaffected by Myo5a at low densities, and was inhibited at high Myo5a densities.

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Long single α-helical tail domains bridge the gap between structure and function of myosin VI

Cited by 115 publications

References 54 publications

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

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

Cargo binding induces dimerization of myosin VI

Plus‐end directed myosins accelerate actin filament sliding by single‐headed myosin VI

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