Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.
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"Unlike other myosins, MVI moves towards the minus (pointed) end of actin filaments (Wells et al. 1999). Inverse directionality of MVI movement, resulting from a difference in the structure of the converter and neck regions, implies its involvement in distinct cellular functions in comparison with other myosins (Elting et al. 2011; Sweeney and Houdusse 2010). "
[Show abstract][Hide abstract] ABSTRACT: Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.
[Show abstract][Hide abstract] ABSTRACT: Myo4p, one of two class V myosins in budding yeast, continuously transports messenger RNA (mRNA) cargo in the cell but is nonprocessive when characterized in vitro. The adapter protein She3p tightly binds to the Myo4p rod, forming a single-headed motor complex. In this paper, we show that two Myo4p-She3p motors are recruited by the tetrameric mRNA-binding protein She2p to form a processive double-headed complex. The binding site for She3p was mapped to a single α helix that protrudes at right angles from She2p. Processive runs of several micrometers on yeast actin-tropomyosin filaments were observed only in the presence of She2p, and, thus, motor activity is regulated by cargo binding. While moving processively, each head steps ~72 nm in a hand-over-hand motion. Coupling two high-duty cycle monomeric motors via a common cargo-binding adapter protein creates a complex with transport properties comparable with a single dimeric processive motor such as vertebrate myosin Va.
The Journal of Cell Biology 11/2011; 195(4):631-41. DOI:10.1083/jcb.201106146 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this, we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use 'hand-over-hand' mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for its diverse cellular functions.