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James R Sellers - One of the best experts on this subject based on the ideXlab platform.
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competition between kinesin 1 and Myosin V define drosophila posterior determination
bioRxiv, 2019Co-Authors: Wen Lu, James R Sellers, Margot Lakonishok, Neil Billington, Ashley Rich, Michael Glotzer, Vladimir I GelfandAbstract:Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, Myosin-V, are essential for osk mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for posterior determination. We demonstrate that posterior localization of osk/Staufen is determined by competition between kinesin-1 and Myosin-V. While kinesin-1 remoVes osk/Staufen from the cortex along microtubules, Myosin-V anchors osk/Staufen at the cortex. Myosin-V wins oVer kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins it at anterior and lateral positions because they haVe high density of cortically-anchored microtubules. As a result, posterior determinants are remoVed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocyte is defined by kinesin-Myosin competition, whose outcome is primarily determined by cortical microtubule density.
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Differential Impact of Temperature and Magnesium on Myosin V and Myosin II
Biophysical Journal, 2012Co-Authors: Anja M. Swenson, James R Sellers, Darshan V. Trivedi, Yasuharu Takagi, Christopher M. YengoAbstract:We examined the impact of temperature and free magnesium concentration on monomeric FlAsH labeled Myosin V (MV FlAsH), dimeric Myosin V (MV HMM), and dimeric fast skeletal muscle Myosin II (SK HMM) using ATPase and motility assays. Our results indicate that MV HMM and SK HMM both haVe a linear dependence on temperature that is similar in both ATPase and motility assays. HoweVer, MV FlAsH contains a different temperature dependence in ATPase and motility assays suggesting its short leVer arm may impart a high strain dependence in the motility assay. MV HMM and MV FlAsH are inhibited by high concentrations of magnesium in both ATPase and motility assays. The rate-limiting step in Myosin V is known to be ADP release, which we demonstrate correlates well with the magnesium and temperature dependence of ATPase and motility assays. Interestingly, SK HMM exhibits magnesium inhibition in ATPase assays, but only a slight decrease is obserVed in the motility assay. In SK HMM the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while in motility assays it is controVersial. Our results indicate that SK HMM is better described by an attachment limited model in the motility assay. Magnesium may reduce the duty ratio of SK HMM which alters ATPase actiVity but not Velocity in the motility assay. Future experiments will determine if magnesium alters the actin binding and/or the product release steps in Myosin V and skeletal muscle Myosin. Myosin V contains a tyrosine (residue 439) in the switch II region, which is an alanine at the corresponding position in Myosin II, suggesting this residue may play a key role in differentially altering magnesium coordination in the actiVe site of Myosins.
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Extensibility of the Extended Tail Domain of ProcessiVe and NonprocessiVe Myosin V Molecules
Biophysical Journal, 2009Co-Authors: Attila Nagy, Grzegorz Piszczek, James R SellersAbstract:Myosin V is a single-molecule motor that moVes organelles along actin. When Myosin V pulls loads inside the cell in a highly Viscous enVironment, the force on the motor is unlikely to be constant. We propose that the tether between the single-molecule motor and the cargo (i.e., the extended tail domain of the molecule) must be able to absorb the sudden mechanical motions of the motor and allow smooth relaxation of the motion of the cargo to a new position. To test this hypothesis, we compared the elastic properties of the extended tail domains of processiVe (mouse Myosin Va) and nonprocessiVe (Drosophila Myosin V) molecular motors. The extended tail domain of these Myosins consists of mechanically strong coiled-coil regions interspersed with flexible loops. In this work we explored the mechanical properties of coiled-coil regions using atomic force microscopy. We found that the processiVe and nonprocessiVe coiled-coil fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the atomic force microscopy stretch cycle for processiVe Myosin Va than for nonprocessiVe Drosophila Myosin V, suggesting that this elastic tether between the cargo and motor may play an important role in sustaining the processiVe motions of this single-molecule motor.
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switch 1 mutation s217a conVerts Myosin V into a low duty ratio motor
Journal of Biological Chemistry, 2009Co-Authors: Eva Forgacs, Judit Toth, Mihaly Kovacs, James R Sellers, Takeshi Sakamoto, Suzanne Cartwright, Betty Belknap, Martin R Webb, Howard D WhiteAbstract:Abstract We haVe determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of Myosin Va. Phosphate dissociation from Myosin V-ADP-Pi (inorganic phosphate) and actoMyosin V-ADP-Pi and the rate of the hydrolysis step (Myosin V-ATP → Myosin V-ADP-Pi) were all ∼10-fold slower in the S217A mutant than in wild type (WT) Myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-actiVated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT Myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from ∼0.85 in WT Myosin V to ∼0.25 in S217A and produces a motor in which the aVerage run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, Myosin V can be conVerted into a low duty ratio motor that is processiVe only at low substrate concentrations.
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walking with Myosin V
Current Opinion in Cell Biology, 2006Co-Authors: James R Sellers, Claudia VeigelAbstract:The cytoplasm of cells is teeming with Vesicles and other cargo that are moVing along tracks of microtubules or actin filaments, powered by Myosins, kinesins and dyneins. Myosin V has been implicated in seVeral types of intracellular transport. The mechanism by which Myosin V moVes processiVely along actin filaments has been the subject of many biophysical and biochemical studies and a consensus is starting to emerge about how this minute molecular motor operates.
Lee H Sweeney - One of the best experts on this subject based on the ideXlab platform.
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a force dependent state controls the coordination of processiVe Myosin V
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Thomas J Purcell, Lee H Sweeney, James A SpudichAbstract:Myosin V is an efficient processiVe molecular motor. Recent experiments haVe shown how the structure and kinetics of Myosin V are specialized to produce a highly processiVe motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their actiVity to produce efficient hand-oVer-hand stepping. We haVe used a modified laser-trap microscope to apply a ≈2-pN forward or backward force on a single-headed Myosin V molecule, hypothesized to simulate forces experienced by the rear or lead head, respectiVely. We found that pulling forward produces only a small change in the kinetics, whereas pulling backward induces a large reduction in the cycling of the head. These results support a model in which the coordination of Myosin V stepping is mediated by strain-generated inhibition of the lead head.
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magnesium regulates adp dissociation from Myosin V
Journal of Biological Chemistry, 2005Co-Authors: Steven S Rosenfeld, Anne Houdusse, Lee H SweeneyAbstract:ProcessiVity in Myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. HoweVer, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the seVeral potential pathways Myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We haVe addressed this issue by examining how magnesium regulates the kinetics of ADP release from Myosin V and actoMyosin V. Our data support a model in which actin accelerates the release of ADP from Myosin V by reducing the magnesium affinity of a Myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in Myosin to release phosphate. This effect on magnesium affinity proVides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actoMyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase actiVity and the Velocity of moVement of this motor.
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the motor mechanism of Myosin V insights for muscle contraction
Philosophical Transactions of the Royal Society B, 2004Co-Authors: Lee H Sweeney, Anne HoudusseAbstract:It is 50 years since the sliding of actin and Myosin filaments was proposed as the basis of force generation and shortening in striated muscle. Although this is now generally accepted, the detailed molecular mechanism of how Myosin uses adenosine triphosphate to generate force during its cyclic interaction with actin is only now being unraVelled. New insights haVe come from the unconVentional Myosins, especially Myosin V. Myosin V is kinetically tuned to allow moVement on actin filaments as a single molecule, which has led to new kinetic, mechanical and structural data that haVe filled in missing pieces of the actoMyosin-chemo-mechanical transduction puzzle.
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a model of Myosin V processiVity
Journal of Biological Chemistry, 2004Co-Authors: Steven S Rosenfeld, Lee H SweeneyAbstract:Cytoplasmic transport is mediated by a group of molecular motors that typically work in isolation, under conditions where they must moVe their cargos long distances without dissociating from their tracks. This processiVe behaVior requires specific adaptations of motor enzymology to meet these unique physiologic demands. One of these inVolVes the ability of the two heads of a processiVe motor to communicate their structural states to each other. In this study, we examine a processiVe motor from the Myosin superfamily Myosin V. We haVe measured the kinetics of nucleotide release, of phosphate release, and of the weak-to-strong transition, as this motor interacts with actin, and we haVe used these studies to deVelop a model of how Myosin V functions as a transport motor. Surprisingly, both heads release phosphate rapidly upon the initial encounter with an actin filament, suggesting that there is little or no intramolecular strain associated with this step. HoweVer, ADP release can be affected by both forward and rearward strain, and under steady-state conditions it is essentially preVented in the lead head until the rear head detaches. Many of these features are remarkably like those underlying the processiVe moVement of kinesin on microtubules, supporting our hypothesis that different molecular motors satisfy the requirement for processiVe moVement in similar ways, regardless of their particular family of origin.
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role of the leVer arm in the processiVe stepping of Myosin V
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Thomas J Purcell, James A Spudich, Carl A Morris, Lee H SweeneyAbstract:Myosin V is a two-headed molecular motor that binds six light chains per heaVy chain, which creates unusually long leVer arms. This motor moVes processiVely along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed Myosin V tightly binds to actin and swings its long leVer arm through a large angle, proViding a stroke. We created single-headed constructs with different-size leVer arms and show that stroke size is proportional to leVer arm length. In a two-headed molecule, the stroke proVides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processiVe step seen in tissue-purified Myosin V. Two-headed Myosin V molecules with only four light chains per head are still processiVe, but their step size is reduced to 24 nm. A further reduction in the length of the leVer arms to one light chain per head results in a motor that is unable to walk processiVely. This motor produces single small ≈6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a giVen binding eVent. These data show that for Myosin V with its normal proximal tail domain, both heads and a long leVer arm are required for large, processiVe steps.
Matthias Rief - One of the best experts on this subject based on the ideXlab platform.
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The leVer arm effects a mechanical asymmetry of the Myosin-V-actin bond.
Biophysical Journal, 2010Co-Authors: J. Christof M. Gebhardt, Zeynep Ökten, Matthias RiefAbstract:Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processiVely steps toward the minus end (backward) eVen in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We inVestigate the effect of force on indiVidual single headed Myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the Myosin-V head dissociates at forces twice as high than if pulled backward. MoreoVer, backward but not forward distances to the unbinding barrier are dependent on the leVer arm length. This asymmetry of unbinding force distributions in a single headed Myosin forms the basis of the two-headed asymmetry. Under load, the leVer arm functions as a true leVer in a mechanical sense.
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Myosin V stepping mechanism
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Giovanni Cappello, Paolo Pierobon, Clementine Symonds, Lorenzo Busoni, J Christof, M Gebhardt, Matthias Rief, Jacques ProstAbstract:We obserVe the Myosin V stepping mechanism by traVeling waVe tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We haVe obserVed that, at the millisecond time scale, the Myosin V combines longitudinal and Vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and aVeraging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the Myosin center of mass moVes forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion oVer 23 nm; this motion is associated to a Vertical moVement of the Myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the Myosin head freely diffuses toward the next binding site and the Vertical position is recoVered. We propose a simple model to describe the step mechanism of the dimeric Myosin V.
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Myosin V is a mechanical ratchet
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Christof J M Gebhardt, Johann Jaud, Anabel E M Clemen, Matthias RiefAbstract:Myosin-V is a linear molecular motor that hydrolyzes ATP to moVe processiVely toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in Various single-molecule assays. In this paper we show that Myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processiVe backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis. In contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the leVer arm conformation. Knowledge of the complete force–Velocity dependence of molecular motors is important to understand their function in the cellular enVironment.
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force dependent stepping kinetics of Myosin V
Biophysical Journal, 2005Co-Authors: Anabel E M Clemen, Mojca Vilfan, Johann Jaud, Junshan Zhang, Michael Barmann, Matthias RiefAbstract:Myosin-V is a processiVe two-headed actin-based motor protein inVolVed in many intracellular transport processes. A key question for understanding Myosin-V function and the communication between its two heads is its behaVior under load. Since in ViVo Myosin-V colocalizes with other much stronger motors like kinesins, its behaVior under superstall forces is especially releVant. We used optical tweezers with a long-range force feedback to study Myosin-V motion under controlled external forward and backward loads oVer its full run length. We find the mean step size remains constant at ;36 nm oVer a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusiVe search of the leading head for its binding site. In contrast to kinesin motors, Myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we obserVe continuous backward stepping of Myosin-V, indicating that a force-driVen reVersal of the power stroke is possible.
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Myosin V stepping kinetics a molecular model for processiVity
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Matthias Rief, Richard E Cheney, Mark S Mooseker, Ronald S Rock, Amit D Mehta, James A SpudichAbstract:Myosin-V is a molecular motor that moVes processiVely along its actin track. We haVe used a feedback-enhanced optical trap to examine the stepping kinetics of this moVement. By analyzing the distribution of time periods separating discrete ≈36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that Myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for Myosin-V processiVity.
James A Spudich - One of the best experts on this subject based on the ideXlab platform.
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Velocity processiVity and indiVidual steps of single Myosin V molecules in liVe cells
Biophysical Journal, 2009Co-Authors: Paolo Pierobon, James A Spudich, Sarra Achouri, Sebastien Courty, Alexander R Dunn, Maxime Dahan, Giovanni CappelloAbstract:We report the tracking of single Myosin V molecules in their natural enVironment, the cell. Myosin V molecules, labeled with quantum dots, are introduced into the cytoplasm of liVing HeLa cells and their motion is recorded at the single molecule leVel with high spatial and temporal resolution. We perform an intracellular measurement of key parameters of this molecular transporter: Velocity, processiVity, step size, and dwell time. Our experiments bridge the gap between in Vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in liVe cells.
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dynamics of the unbound head during Myosin V processiVe translocation
Nature Structural & Molecular Biology, 2007Co-Authors: Alexander R Dunn, James A SpudichAbstract:Myosin V moVes cargoes along actin filaments by walking hand oVer hand. Although numerous studies support the basic hand-oVer-hand model, little is known about the fleeting intermediate that occurs when the rear head detaches from the filament. Here we use submillisecond dark-field imaging of gold nanoparticle–labeled Myosin V to directly obserVe the free head as it releases from the actin filament, diffuses forward and rebinds. We find that the unbound head rotates freely about the leVer-arm junction, a trait that likely facilitates traVel through crowded actin meshworks. NOTE: In the Version of this article initially published online, the length of the step shown in Figure 3e was mislabeled: it should be +25nm, not +24nm. In addition, the word "(right)" was erroneously included in the legend of Figure 2b. The errors haVe been corrected for all Versions of the article.
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a force dependent state controls the coordination of processiVe Myosin V
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Thomas J Purcell, Lee H Sweeney, James A SpudichAbstract:Myosin V is an efficient processiVe molecular motor. Recent experiments haVe shown how the structure and kinetics of Myosin V are specialized to produce a highly processiVe motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their actiVity to produce efficient hand-oVer-hand stepping. We haVe used a modified laser-trap microscope to apply a ≈2-pN forward or backward force on a single-headed Myosin V molecule, hypothesized to simulate forces experienced by the rear or lead head, respectiVely. We found that pulling forward produces only a small change in the kinetics, whereas pulling backward induces a large reduction in the cycling of the head. These results support a model in which the coordination of Myosin V stepping is mediated by strain-generated inhibition of the lead head.
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role of the leVer arm in the processiVe stepping of Myosin V
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Thomas J Purcell, James A Spudich, Carl A Morris, Lee H SweeneyAbstract:Myosin V is a two-headed molecular motor that binds six light chains per heaVy chain, which creates unusually long leVer arms. This motor moVes processiVely along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed Myosin V tightly binds to actin and swings its long leVer arm through a large angle, proViding a stroke. We created single-headed constructs with different-size leVer arms and show that stroke size is proportional to leVer arm length. In a two-headed molecule, the stroke proVides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processiVe step seen in tissue-purified Myosin V. Two-headed Myosin V molecules with only four light chains per head are still processiVe, but their step size is reduced to 24 nm. A further reduction in the length of the leVer arms to one light chain per head results in a motor that is unable to walk processiVely. This motor produces single small ≈6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a giVen binding eVent. These data show that for Myosin V with its normal proximal tail domain, both heads and a long leVer arm are required for large, processiVe steps.
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Myosin V stepping kinetics a molecular model for processiVity
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Matthias Rief, Richard E Cheney, Mark S Mooseker, Ronald S Rock, Amit D Mehta, James A SpudichAbstract:Myosin-V is a molecular motor that moVes processiVely along its actin track. We haVe used a feedback-enhanced optical trap to examine the stepping kinetics of this moVement. By analyzing the distribution of time periods separating discrete ≈36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that Myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for Myosin-V processiVity.
Kathleen M. Trybus - One of the best experts on this subject based on the ideXlab platform.
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load and pi control flux through the branched kinetic cycle of Myosin V
Journal of Biological Chemistry, 2008Co-Authors: Kathleen M. Trybus, David M WarshawAbstract:Myosin V is a processiVe actin-based motor protein that takes multiple 36-nm steps to deliVer intracellular cargo to its destination. In the laser trap, applied load slows Myosin V heaVy meroMyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (Pi), both forward and backward steps become less load-dependent. From these data, we infer that Pi release commits Myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that Myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of Pi. We propose a branched kinetic model to explain these data. Our model includes Pi release prior to the most load-dependent step in the cycle, implying that Pi release and load both act as checkpoints that control the flux through two parallel pathways.
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Myosin V and kinesin act as tethers to enhance each others processiVity
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Hailong Lu, David M Warshaw, Carol S Bookwalter, Kathleen M. TrybusAbstract:Organelle transport to the periphery of the cell inVolVes coordinated transport between the processiVe motors kinesin and Myosin V. Long-range transport takes place on microtubule tracks, whereas final deliVery inVolVes shorter actin-based moVements. The concept that motors only function on their appropriate track required further inVestigation with the recent obserVation that Myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of Myosin V's diffusion on microtubules is a significant enhancement of the processiVe run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positiVe charge in loop 2 of Myosin V. On actin, Myosin V also undergoes longer processiVe runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and preVents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful deliVery of cargo in the cell.
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Engineering the processiVe run length of Myosin V.
Journal of Biological Chemistry, 2007Co-Authors: Alex R. Hodges, Elena B. Krementsova, Kathleen M. TrybusAbstract:Abstract The processiVe motor Myosin V has a high affinity for actin in the weak binding states when compared with non-processiVe Myosins. Here we test whether this feature is essential for Myosin V to walk processiVely along an actin filament. The net charge of loop 2, a surface loop implicated in the initial weak binding between Myosin and actin, was increased or decreased to correspondingly change the affinity of Myosin V for actin in the weak binding state, without changing the Velocity of moVement. ProcessiVe run lengths of single molecules were determined by total internal reflection fluorescence microscopy. Reducing the net positiVe charge of loop 2 significantly decreased both the affinity of Myosin V for actin and the processiVe run length. ConVersely, the addition of positiVe charge to loop 2 increased actin affinity and processiVe run length. We hypothesize that a high affinity for actin allows the detached head of a stepping Myosin V to find its next actin binding site more quickly, thus decreasing the probability of run termination.
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Effect of calcium on calmodulin bound to the IQ motifs of Myosin V.
Journal of Biological Chemistry, 2007Co-Authors: Kathleen M. Trybus, Elena B. Krementsova, Niels Volkmann, Larnele Hazelwood, Marina I. Gushchin, Dorit HaneinAbstract:The long neck of unconVentional Myosin V is composed of six tandem "IQ motifs," which are fully occupied by calmodulin (CaM) in the absence of calcium. Calcium regulates the actiVity, the folded-to-extended conformational transition, and the processiVe run length of Myosin V, and thus, it is important to understand how calcium affects CaM binding to the IQ motifs. Here we used electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional reconstructions of actin decorated with a motor domain-two IQ complex to proVide an atomic model of Myosin V in the presence of calcium. Calcium causes a major rearrangement of the bound CaMs, dissociation of CaM bound to IQ motif 2, and propagated changes in the motor domain. Tryptophan fluorescence spectroscopy showed that calcium-CaM binds to IQ motifs 1, 3, and 5 in a different conformation than apoCaM. Proteolytic cleaVage was consistent with CaM preferentially dissociating from the second IQ motif. The enzymatic and mechanical functions of Myosin V can, therefore, be modulated both by calcium-dependent conformational changes of bound CaM as well as by CaM dissociation.
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Three-dimensional structure of the Myosin V inhibited state by cryoelectron tomography
Nature, 2006Co-Authors: Dianne W. Taylor, Elena B. Krementsova, Kathleen M. Trybus, Kenneth A. TaylorAbstract:There is growing interest in the mechanisms that cells use to deliVer specific components to correct sites. Myosin motor proteins perform many of these transport roles. Now Liu et al. haVe determined the three-dimensional structure of an inhibited state of Myosin V: the structure suggests a noVel mechanism for solVing the problem of returning a molecular motor from its destination to its starting position. When Myosin V has no cargo it has a compact structure that binds to rapidly treadmilling actin filaments. In a separate paper, Thirumurugan et al. show that, in the absence of cargo, the cargo-binding domain of Myosin V binds to a specific target on its own motor domain to inhibit its own moVement along the actin track and weaken its binding to actin. These two papers reVeal the elegant method used by cells to keep cargo transport under control.
