Quantized velocities at low myosin densities in an in vitro motility

Uyeda, Taro Q.P.; Warrick, Hans M.; Kron, Stephen J.; Spudich, James A.

doi:10.1038/352307a0

Cited by 180 publications

(113 citation statements)

References 18 publications

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“…1G). In the case that the duty ratio-the fraction of binding time over the single ATPase turnover-of the motor is low, it is generally considered that the average speed should decrease as the density of the motor decreases (18,19). This model coincides with our observations.…”

Section: Discussionsupporting

confidence: 81%

Unitary step of gliding machinery in Mycoplasma mobile

Kinosita

Nakane

Sugawa

et al. 2014

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

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Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 μm·s −1 with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

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Section: Discussionsupporting

confidence: 81%

Unitary step of gliding machinery in Mycoplasma mobile

Kinosita

Nakane

Sugawa

et al. 2014

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

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“…Due to very low affinity for actin, L3(-5) could not move actin filaments in the standard buffer for in vitro motility assay. However, in the presence of 0.8% methylcellulose that prevent detachment of actin filaments from the myosincoated surface (30), the velocity of L3(-5) could be observed, and it was 70% that of the wild type (Table 3).…”

Section: Loop 3 Mutantsmentioning

confidence: 99%

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.

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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

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“…This behavior is in marked contrast to the myosin II molecules that power muscle contrac-tion. These low duty ratio myosins are designed to make single transient interactions with actin and spend most of their kinetic cycle dissociated from actin (10,11).…”

mentioning

confidence: 99%

Neck Length and Processivity of Myosin V

Sakamoto

Wang

Schmitz³

et al. 2003

Journal of Biological Chemistry

147

146

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Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 M or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1 molecules bound to an ␣-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4). The six IQ motifs of all myosin V superfamily members are separated by 25-23-25-23-25 amino acids.The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpede...

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Quantized velocities at low myosin densities in an in vitro motility

Cited by 180 publications

References 18 publications

Unitary step of gliding machinery in Mycoplasma mobile

Unitary step of gliding machinery in Mycoplasma mobile

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

Neck Length and Processivity of Myosin V

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