Simultaneous tracking of two motor domains reveals near simultaneous steps and stutter steps of myosin 10 on actin filament bundles
Introduction
The myosin superfamily of molecular motors provides a large number of functions that involve movement on actin filaments in cells, including cytokinesis, intracellular organelle movements, powering muscle contraction, movement of cells themselves, and other functions [[1], [2], [3], [4], [5]]. There are at least 79 classes of myosin within the myosin superfamily [3] that follow a basic blueprint of a N-terminal motor and a C-terminal targeting (cargo-binding) domain. In dimer-forming myosins, the C-terminal targeting domain is preceded by a coiled coil. The motor domain ends with the converter subdomain, which amplifies rearrangements in the motor and is the first component of the myosin lever arm. The lever arm contains a variable number of CaM/light chain binding sites and additional extensions are found in some myosin classes.
A number of dimeric myosins can move processively on actin, taking hand-over-hand steps, including Class V (Myo5), Class VI (Myo6), and Class X (Myo10), but with different stepping behaviors [6,7]. What is quite different among these myosins is the design of their “lever arms”, which are extensions from the motor domain that amplify changes in the motor conformation to produce a powerstroke on actin. Unlike the lever arm of Myo5, the lever arm of Myo10 is made up of a combination of IQ motifs with associated light chains and a stable, single alpha-helical (SAH) domain.
Myo10 is involved in the formation of and trafficking in filopodia [1]. The formation of filopodia was reported to be induced by the dimerization of Myo10 [8]. There has been controversy as to whether Myo10 preferentially traffics on actin bundles, and if so, what the nature of the stepping behavior is [6,7,9,10]. This was the result of Myo10 having an anti-parallel coiled coil, unlike other myosin classes [11,12]. Proper dimerization is important for normal function of Myo10, allowing its movement to the tips of filopodia and its proper localization in cells [8,11,13]. However, the stepping mechanism of Myo10 containing the anti-parallel coiled coil is not clearly understood. Herein, we performed in vitro single-molecule motility assay of Myo10 using two-color tracking of two heads of Myo10 that allowed us to independently follow the positions of both heads of Myo10 dimers during stepping on actin bundles.
Section snippets
Myo10 constructs, expression, purification, and labelling
Full-length human Myo10 was constructed with either mApple (red fluorescence protein) or a HaloTag added to the N-terminus, which were preceded by an N-terminal Flag tag. These constructs were used to create recombinant baculoviruses for expression in SF9 cells [12], similar to the previously described method for Myo5 [14]. The virus coding for mApple was co-infected with the virus coding for HaloTag at a ratio of 1:1 in SF9 cells. Three days after infection with these constructs, SF9 cells
Labeling two heads of full-length Myo10 with different fluorophores
To label both heads of full-length Myo10 dimers with two fluorophores, we generated two constructs for Myo10. One construct contained HaloTag fused to the N-terminus of full-length Myo10 (Fig. 1A), and the other construct contained mApple (red fluorescence protein) at the N-terminus(Fig. 1A), which we previously generated and characterized [12]. To promote hetero-dimerization of full-length Myo10 labeled with mApple and a HaloTag conjugated with Alexa 488 (as depicted in Fig. 1B), expressed
Discussion
What was surprising in our results is that Myo10, unlike any processive myosin previously described, frequently takes near simultaneous steps and occasionally one head takes multiple steps, or stutter steps, rather than the normal hand-over-hand stepping. Near simultaneous steps (steps of near zero dwell time) have not been observed for other processive myosins, such as Myo5 and Myo6. Such stepping would be prevented by “gating” of the heads. Gating manifests as a stalling of the powerstroke
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
We thank Chun Sum Brian Pang for help with data analysis. This work was supported by grants from the Research Grants Council of Hong Kong (26101117, 16101518, N_HKUST613/17, and A-HKUST603/17 to H.P.) and the Innovation and Technology Commission (ITCPD/17-9 to H.P.). HLS was supported by NIH grant DC009100.
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H.Y., H.C.M.C. and Q.Q.N. contributed equally to this work.