Engineering the Processive Run Length of Myosin V

Hodges, Alex R.; Krementsova, Elena B.; Trybus, Kathleen M.

doi:10.1074/jbc.m703968200

Cited by 43 publications

(56 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At high dilutions, actin filaments could be observed pivoting around a single attachment point while they were moving, and they dissociated when the end of the filament passed the attachment point. Quantum dots associated with maximally a few Myo9-head molecules were observed to move along actin filaments over considerable distances with an average run length of 3.1 m. This run length is comparable with that of the dimeric processive motor myosin Va (47). Continuous movement for such long distances implicates that motility is driven by a coordinated mechanism to prevent detachment and not by independently bound motor molecules.…”

Section: Discussionmentioning

confidence: 60%

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Liao¹,

Elfrink²,

Bähler³

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Mammalian myosin IXb (Myo9b) has been shown to exhibit unique motor properties in that it is a single-headed processive motor and the rate-limiting step in its chemical cycle is ATP hydrolysis. Furthermore, it has been reported to move toward the minus-and the plus-end of actin filaments. To analyze the contribution of the light chain-binding domain to the movement, processivity, and directionality of a single-headed processive myosin, we expressed constructs of Caenorhabditis elegans myosin IX (Myo9) containing either the head (Myo9-head) or the head and the light chain-binding domain (Myo9-head-4IQ). Both constructs supported actin filament gliding and moved toward the plus-end of actin filaments. We identified in the head of class IX myosins a calmodulin-binding site at the N terminus of loop 2 that is unique among the myosin superfamily members. Ca 2؉ /calmodulin negatively regulated ATPase and motility of the Myo9-head. The Myo9-head demonstrated characteristics of a processive motor in that it supported actin filament gliding and pivoting at low motor densities. Quantum dot-labeled Myo9-head moved along actin filaments with a considerable run length and frequently paused without dissociating even in the presence of obstacles. We conclude that class IX myosins are plus-end-directed motors and that even a single head exhibits characteristics of a processive motor.Myosins form a large superfamily of actin-based molecular motors that is composed of at least 35 classes (1). Class IX myosins arose in metazoa after the separation of the fungi (1). Invertebrates contain a single myosin class IX gene with the exception of the Drosophila species that have lost their class IX myosin. Bony fishes contain four myosin IX genes and other vertebrates, including mammalia two genes. The two class IX myosins in mammals, Myo9a 2 and Myo9b, exist in multiple splice variants (2). Myo9a has been shown to play a role in epithelial differentiation and morphology whereas Myo9b regulates the migration of macrophages and possibly other immune cells (3, 4). Class IX myosins share a similar structure with the myosins of the other classes, containing a head region, a calmodulin/light chain-binding domain, and a tail region.Additionally, class IX myosins carry some unique features, including a large N-terminal extension preceding the head domain and a long insertion within the head domain in loop 2. The tail region comprises a C1 zinc-binding domain and a RhoGAP domain. Because of this RhoGAP domain, class IX myosins are involved in signal transduction regulating the dynamics of the actin cytoskeleton (2, 5).Mammalian Myo9b, the only class IX myosin studied so far in vitro, exhibits unique mechano-chemical properties. It has been reported to take multiple successive steps along actin filaments without dissociating, indicating that it is a processive motor (6 -8). This is remarkable because Myo9b is a singleheaded myosin. Other myosins that move processively on actin filaments, such as myosin V, dimeric myosin VI, and myosin VII, are two-...

show abstract

Section: Discussionmentioning

confidence: 60%

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Liao¹,

Elfrink²,

Bähler³

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The inverse correlation between the net charge of loop 2 and the velocity may be a general rule for myosin velocity although an exceptional result was reported for slow myosin with very long loop 2. Addition of positive charges to loop 2 of myosin V did not alter the velocity (29). Since ␣-helix after loop 2 is connected to a ␤-strand that surrounds the nucleotide binding site of myosin (36), strong interaction with actin through loop 2 somehow reduces the ADP release rate and thus velocity.…”

Section: Discussionmentioning

confidence: 99%

“…Most myosins have positively charged a loop 2 with a cluster of lysine residues (Table 1). Many studies using various myosins suggested that the net charge of loop 2 controls the affinity of myosin for actin (25)(26)(27)(28)(29). Unlike other myosins, loop 2 of Chara myosin is very short and the Fig.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Ito

Yamaguchi

Yanase

et al. 2009

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

View full text Add to dashboard Cite

Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected Vmax and Kapp of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected Kapp and the velocity, although it did not affect Vmax. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.actin ͉ ATPase ͉ motility ͉ cytoplasmic streaming ͉ molecular engineering C ytoplasmic streaming in characean algal cells is extremely fast, and this streaming is brought about by the movement of myosin-coated organelles along actin filament bundles fixed inside the cell (1). Myosin purified from Chara corallina could translocate actin filaments in the in vitro motility assay at a velocity comparable to that of the cytoplasmic streaming (approximately 50 m s Ϫ1 ) (2, 3). This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5, 6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length. Relevant steps in actomyosin ATPase cycle to the sliding velocity are ADP release from actomyosin and ATP reassociation resulting in actin-myosin dissociation. Kinetic analyses of Chara myosin motor domain revealed that both the ADP release and the ATP-induced dissociation from actin were very fast (6). Time spent in the strongly bound state with actin, which was estimated from these rates, was less than 1 ms. This very short strongly bound state, together with a large step size (7), are probably the reason for the very high velocity of Chara myosin. Actin-activated ATPase activity of expressed Chara motor domain was approximately 500 s Ϫ1 head Ϫ1

show abstract

“…2C). The decay constant was determined as previously described (21,22) by using an exponential fit of the cumulative probability distribution of the data (see Fig. S3A), as indicated in Fig.…”

Section: Resultsmentioning

confidence: 99%

Cargo binding induces dimerization of myosin VI

Phichith

Travaglia²,

Yang³

et al. 2009

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

105

104

View full text Add to dashboard Cite

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...

show abstract

Engineering the Processive Run Length of Myosin V

Cited by 43 publications

References 34 publications

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Cargo binding induces dimerization of myosin VI

Contact Info

Product

Resources

About