Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach

Mukherjea, Monalisa; Llinas, P.; Kim, Hyeong Jun; Travaglia, Mirko; Safer, Daniel; Menetrey, J.; Franzini‐Armstrong, Clara; Selvin, Paul R.; Houdusse, Anne; Sweeney, H. Lee

doi:10.1016/j.molcel.2009.07.010

Cited by 91 publications

(206 citation statements)

References 42 publications

(80 reference statements)

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“…2 B and D). This stepping pattern is believed to be a property of myoVI's inherent flexibility Step Size (nm) within the tail that allows the motor to take such long steps with short lever arms (22). The forward step lifetime of 222 AE 24 ms (N ¼ 110) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Safer³

et al. 2011

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

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Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.intracellular transport | motility assay | processivity | single molecule biophysics I ntracellular cargo transport relies on at least two classes of myosin molecular motors to navigate the actin cytoskeleton. Class V (myoV) and class VI (myoVI) myosins are double-headed motors that transport cargo in opposite directions along polarized actin filament tracks (1, 2). These motors convert the energy of ATP hydrolysis into force and motion and in doing so step processively on actin in a hand-over-hand fashion. With the plusends of actin filaments oriented toward the cell periphery, myoV transport contributes to exocytosis, whereas myoVI transport is critical to endocytosis (1, 2). Ensembles of molecular motors share the responsibility for transporting a single vesicle. For example, approximately 60 myoV motors coat a melanosome, although a smaller number is likely to be engaged with the track at any given time (3). Transport can also be bidirectional when oppositely directed motors are operative, as for axonal transport by the microtubule-based motors, kinesin and dynein (4, 5). With myoV and myoVI colocalizing to organelles (6, 7), these motors may engage in a virtual tug of war. If so, how do these oppositely directed myosin motors mechanically interact so that cargo is efficiently delivered to its destination?Individual motors within a transport ensemble may coordinate, cooperate, or mechanically impede one another, but determining by which mode they operate is complicated by the cargo geometry, the number and directionality of engaged motors, and the physical challenges presented by the dense actin cytoskeleton. Therefore, investigators have designed in vitro experiments to limit the number of coupled motors so that mechanistic modeling efforts were tractable (8-11). For these models, the velocity and direction of cargo transport generated by ensembles of antagonistic motors depended solely on the relative number of motors pulling in either direction (12, 13). The mechanical interactions between motors and the resultant stepping dynamics of each motor within the ensemble were beyond the predictive capacity of these models. Therefore, ...

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Section: Resultsmentioning

confidence: 99%

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Safer³

et al. 2011

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

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“…Generally, these regulated forms are considered undesirable in mechanical studies, so tail truncations or substitutions are employed to activate the motors. However, note that our GCN4 dimerization domain is insufficient to force dimerization under the extremely low concentrations used here (28,31). Therefore, we know that additional regions of the native myosin X sequence must contribute to the dimerization as well.…”

Section: Structured Tail Domain Is Responsible For Bundle Selection Inmentioning

confidence: 90%

“…In certain cases, cargo binding regulates dimerization and function. This form of regulation may be general in cells, as it prevents myosins from engaging tracks and accumulating at their final destination without their cargoes (31). Moreover, myosin activity can be regulated by direct headto-tail interactions in the absence of cargo (31,32).…”

Section: Structured Tail Domain Is Responsible For Bundle Selection Inmentioning

confidence: 99%

“…This form of regulation may be general in cells, as it prevents myosins from engaging tracks and accumulating at their final destination without their cargoes (31). Moreover, myosin activity can be regulated by direct headto-tail interactions in the absence of cargo (31,32). Generally, these regulated forms are considered undesirable in mechanical studies, so tail truncations or substitutions are employed to activate the motors.…”

Section: Structured Tail Domain Is Responsible For Bundle Selection Inmentioning

confidence: 99%

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Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Nagy

Rock

2010

Journal of Biological Chemistry

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Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses.The essential role of cytoskeletal motor proteins in organizing cellular compartments and cargoes is widely accepted (1). However, many of the molecular details of the overall transport and organization process are poorly understood. These essential features include how motors engage cargoes at their source, how motors are activated once they engage cargo, how they choose the correct set of cytoskeletal tracks, how they disengage from cargo at their destination, and how they return to sources of cargo. Clearly, errors at any of these stages would lead to faults in cellular organization and misplaced cargoes.To begin to address these questions we have focused on a particular motor protein, myosin X, due to its ability to navigate to a precise location within the cell (2). Myosin X is found at the mitotic and meiotic spindle, where it sets the orientation of the spindle relative to the substrate and influences spindle length (3, 4). However, myosin X is more commonly found in interphase cells at the tips of filopodia (5). These long, slender projections at the leading edge of migrating cells are involved in cell motility and environmental sensing. Myosin X transports components such as Ena/VASP and integrins that are found in the filopodial tip complex (2, 6). The early work by Berg et al. (5) demonstrated that myosin X reaches filopodial tips under its own power. Recently, we found that myosin X identifies filopodia by recognizing the fascin-bundled actin filaments found in the filopodial core (7). Myosin X takes long processive runs along these tightly bundled actin filaments while largely ignoring isolated actin filaments found throughout the cell. In contrast, myosin V does not distinguish between single actin filaments and bundled actin filaments when taking processive runs.Here, we seek to identify the "selectivity module" that allows myosin X to recognize bundled actin filaments. Our first approach is to swap similar domains between the selective myosin X and the nonselective myosin V. In this approach, we are aided by the fact that both m...

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“…For c.897G4T, as exon 10 codes for an essential region of the motor, it may be that the protein by the mutant allele produced is non-functional and unstable, resulting in haploinsufficiency for myosin VI in these patients. The p.L926Q variant is located in a region rich in charged residues, which has been proposed to form a single alpha helix (SAH) and is also a potential proximal dimerization region, in the tail region of the myosin VI protein (http://www.uniprot.org/uniprot/) 40 (Figure 1f). A mutation in this region, which has been proposed to form a SAH in the monomeric motor molecule, should not have a drastic effect on the monomeric motor.…”

Section: Discussionmentioning

confidence: 99%

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

et al. 2013

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Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

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Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach

Cited by 91 publications

References 42 publications

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

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