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Tuning myosin-driven sorting on cellular actin networks.

Hariadi RF, Sommese RF, Sivaramakrishnan S - Elife (2015)

Bottom Line: While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm.This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold.Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States.

ABSTRACT
Myosin V and VI are antagonistic motors that cohabit membrane vesicles in cells. A systematic study of their collective function, however, is lacking and forms the focus of this study. We functionally reconstitute a two-dimensional actin-myosin interface using myosin V and VI precisely patterned on DNA nanostructures, in combination with a model keratocyte actin meshwork. While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm. This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold. Pairing computation with experimental observations suggests that the ratio of motor stall forces is a key determinant of the observed competitive outcomes. Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type.

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Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.(A–C) Kymographs showing the movement of indicated motor ensembles along actin filaments. Scaffolds with myosin V and VI display unidirectional movements toward plus-or minus-ends of the actin filament. The gray hexagon represents the organization of attachment sites on the scaffold, the red and blue arrows denote myosin V and VI, respectively. (D) Speed of plus-end (blue) and minus-end (red) directed movement of indicated scaffolds on actin filaments. Error bars are S.E.M. (E) Relative frequency of plus-end (n+) and minus-end (n−) directed movement for 1V:1VI scaffolds on actin filaments and keratocyte actin networks. Outward flux (Φout) is defined as the fraction of plus-end directed trajectories. Error bars are S.E.M. and were generated by bootstrapping (N ≥ 202 trajectories; ≥3 experiments).DOI:http://dx.doi.org/10.7554/eLife.05472.006
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fig2: Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.(A–C) Kymographs showing the movement of indicated motor ensembles along actin filaments. Scaffolds with myosin V and VI display unidirectional movements toward plus-or minus-ends of the actin filament. The gray hexagon represents the organization of attachment sites on the scaffold, the red and blue arrows denote myosin V and VI, respectively. (D) Speed of plus-end (blue) and minus-end (red) directed movement of indicated scaffolds on actin filaments. Error bars are S.E.M. (E) Relative frequency of plus-end (n+) and minus-end (n−) directed movement for 1V:1VI scaffolds on actin filaments and keratocyte actin networks. Outward flux (Φout) is defined as the fraction of plus-end directed trajectories. Error bars are S.E.M. and were generated by bootstrapping (N ≥ 202 trajectories; ≥3 experiments).DOI:http://dx.doi.org/10.7554/eLife.05472.006

Mentions: Two previous reports suggest that equal numbers of myosin V and VI anchored to the same scaffold display solely unidirectional movement (Ali et al., 2011; Hariadi et al., 2014). However, they disagree in the observed outcome of the competition. Myosin V dominates the competition (79%) when it is tethered to myosin VI through a quantum dot (2 total) and the two compete on a single actin filament (Ali et al., 2011). In contrast, myosin V and VI are evenly matched (myosin V wins 52%) when two of each motor (4 total) are tethered to a DNA nanostructure and they compete on a two-dimensional cellular actin network. This discrepancy between the observations could stem from either the scaffold type (quantum dot vs DNA nanostructure), the total motor number (2 vs 4), or the actin architecture (single filament vs keratocyte-derived actin network). We first tested the influence of scaffold type by assessing the competition between a single myosin V dimer and a single myosin VI dimer on 1D actin filaments (Figure 2). In positive controls, ØV:2VI scaffolds (Figure 2A) move toward the minus-end of the actin filaments, whereas 2V:ØVI scaffolds (Figure 2C) travel toward the plus-end. Consistent with previous reports (Ali et al., 2011; Hariadi et al., 2014), scaffolds with both myosin V and myosin VI (1V:1VI) commit to a single direction on actin filaments (>99%; Figure 2B) with no directional reversal detected. The movement of 1V:1VI scaffolds on single actin filaments is dominated by plus-end directed movement (Φout = 68 ± 1%; Figure 2E), which is qualitatively consistent with previous observations using quantum dot scaffolds (79% plus-end directed [Ali et al., 2011]). Hence, scaffold type (quantum dot vs DNA nanostructure) is not the key determinant of competitive outcome. We next examined the influence of actin architecture. In contrast to single actin filaments, both plus and minus-end directed movement is equally represented (Φout = 52 ± 1%; Figure 2E) for 1V:1VI scaffolds moving along 2D keratocyte actin networks. Hence, the discrepancy between previous reports using quantum dots (Ali et al., 2011) and DNA nanostructures (Hariadi et al., 2014) stems primarily from the actin architecture.10.7554/eLife.05472.006Figure 2.Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.


Tuning myosin-driven sorting on cellular actin networks.

Hariadi RF, Sommese RF, Sivaramakrishnan S - Elife (2015)

Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.(A–C) Kymographs showing the movement of indicated motor ensembles along actin filaments. Scaffolds with myosin V and VI display unidirectional movements toward plus-or minus-ends of the actin filament. The gray hexagon represents the organization of attachment sites on the scaffold, the red and blue arrows denote myosin V and VI, respectively. (D) Speed of plus-end (blue) and minus-end (red) directed movement of indicated scaffolds on actin filaments. Error bars are S.E.M. (E) Relative frequency of plus-end (n+) and minus-end (n−) directed movement for 1V:1VI scaffolds on actin filaments and keratocyte actin networks. Outward flux (Φout) is defined as the fraction of plus-end directed trajectories. Error bars are S.E.M. and were generated by bootstrapping (N ≥ 202 trajectories; ≥3 experiments).DOI:http://dx.doi.org/10.7554/eLife.05472.006
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fig2: Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.(A–C) Kymographs showing the movement of indicated motor ensembles along actin filaments. Scaffolds with myosin V and VI display unidirectional movements toward plus-or minus-ends of the actin filament. The gray hexagon represents the organization of attachment sites on the scaffold, the red and blue arrows denote myosin V and VI, respectively. (D) Speed of plus-end (blue) and minus-end (red) directed movement of indicated scaffolds on actin filaments. Error bars are S.E.M. (E) Relative frequency of plus-end (n+) and minus-end (n−) directed movement for 1V:1VI scaffolds on actin filaments and keratocyte actin networks. Outward flux (Φout) is defined as the fraction of plus-end directed trajectories. Error bars are S.E.M. and were generated by bootstrapping (N ≥ 202 trajectories; ≥3 experiments).DOI:http://dx.doi.org/10.7554/eLife.05472.006
Mentions: Two previous reports suggest that equal numbers of myosin V and VI anchored to the same scaffold display solely unidirectional movement (Ali et al., 2011; Hariadi et al., 2014). However, they disagree in the observed outcome of the competition. Myosin V dominates the competition (79%) when it is tethered to myosin VI through a quantum dot (2 total) and the two compete on a single actin filament (Ali et al., 2011). In contrast, myosin V and VI are evenly matched (myosin V wins 52%) when two of each motor (4 total) are tethered to a DNA nanostructure and they compete on a two-dimensional cellular actin network. This discrepancy between the observations could stem from either the scaffold type (quantum dot vs DNA nanostructure), the total motor number (2 vs 4), or the actin architecture (single filament vs keratocyte-derived actin network). We first tested the influence of scaffold type by assessing the competition between a single myosin V dimer and a single myosin VI dimer on 1D actin filaments (Figure 2). In positive controls, ØV:2VI scaffolds (Figure 2A) move toward the minus-end of the actin filaments, whereas 2V:ØVI scaffolds (Figure 2C) travel toward the plus-end. Consistent with previous reports (Ali et al., 2011; Hariadi et al., 2014), scaffolds with both myosin V and myosin VI (1V:1VI) commit to a single direction on actin filaments (>99%; Figure 2B) with no directional reversal detected. The movement of 1V:1VI scaffolds on single actin filaments is dominated by plus-end directed movement (Φout = 68 ± 1%; Figure 2E), which is qualitatively consistent with previous observations using quantum dot scaffolds (79% plus-end directed [Ali et al., 2011]). Hence, scaffold type (quantum dot vs DNA nanostructure) is not the key determinant of competitive outcome. We next examined the influence of actin architecture. In contrast to single actin filaments, both plus and minus-end directed movement is equally represented (Φout = 52 ± 1%; Figure 2E) for 1V:1VI scaffolds moving along 2D keratocyte actin networks. Hence, the discrepancy between previous reports using quantum dots (Ali et al., 2011) and DNA nanostructures (Hariadi et al., 2014) stems primarily from the actin architecture.10.7554/eLife.05472.006Figure 2.Unidirectional movement and sorting of scaffolds with myosin V and VI along single actin filaments.

Bottom Line: While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm.This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold.Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States.

ABSTRACT
Myosin V and VI are antagonistic motors that cohabit membrane vesicles in cells. A systematic study of their collective function, however, is lacking and forms the focus of this study. We functionally reconstitute a two-dimensional actin-myosin interface using myosin V and VI precisely patterned on DNA nanostructures, in combination with a model keratocyte actin meshwork. While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm. This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold. Pairing computation with experimental observations suggests that the ratio of motor stall forces is a key determinant of the observed competitive outcomes. Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type.

Show MeSH
Related in: MedlinePlus