Ca2+ Causes Release of Myosin Heads from the Thick Filament Surface on the Milliseconds Time Scale

Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue N, Worcester, MA 01655-0106, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 04/2003; 327(1):145-58. DOI: 10.1016/S0022-2836(03)00098-6
Source: PubMed


We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca2+. Negative staining reveals that when Ca2+ binds to the heads of relaxed Ca2+ -regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca2+. The structural change is thus a result of Ca2+ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca2+ -binding. We therefore used a time-resolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca2+ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca2+ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction.

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    • "The results of investigation on the functionality of skeletal muscle myosin bio-nanorobot showed that the steps size was fairly uniform with an average size of 11 nm under conditions of low load [22]. Muscle contraction process is also regulated by Ca 2+ ions [23]. At low Ca 2+ levels, actin-myosin interaction is inhibited, and muscle is at rest. "
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    ABSTRACT: Kinesin and muscle myosin are considered as physical bio-nanoagents able to sense their cells through their sensors, make decision internally, and perform actions through their actuators. This paper has investigated and compared the flexible (reactive, pro-active, and interactive) autonomous behaviors of kinesin and muscle myosin bio-nanorobots. Using an automata algorithm, the agent-based deterministic finite automaton models of the internal decision making processes of the bio-nanorobots (as their reactive and pro-active capabilities) were converted to their respective computational regular languages (as their interactive capabilities). The resulted computational languages could represent the flexible autonomous behaviors of the bio-nanorobots. The proposed regular languages also reflected the degree of the autonomy and intelligence of internal decision-making processes of the bio-nanorobots in response to their environments. The comparison of flexible autonomous behaviors of kinesin and muscle myosin bio-nanorobots indicated that both bio-nanorobots employed regular languages to interact with their environments through two sensors and one actuator. Moreover, the results showed that kinesin bio-nanorobot used a more complex regular language to interact with its environment compared with muscle myosin bio-nanorobot. Therefore, our results have revealed that the flexible autonomous behavior of kinesin bio-nanorobot was more complicated than the flexible autonomous behavior of muscle myosin bio-nanorobot.
    Full-text · Article · Nov 2013 · IEEE Transactions on Industrial Electronics
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    • "In the inactive (relaxed) state of muscle, the heads of both regulated and unregulated myosin filaments are ordered, turn over ATP slowly, and undergo minimal interaction with actin filaments (Lymn and Taylor, 1971; Xu et al., 1996; Craig and Woodhead, 2006). Activation leads to disordering of the head array (Huxley and Brown, 1967; Zhao and Craig, 2003), increase in ATPase activity, and interaction with actin. "
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    ABSTRACT: Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II-based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head-head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head-head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the 'blocked' myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head-head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II-based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.
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    • "We have used a procedure of incubating filaments on the EM grid with F-actin buffer (containing 0.2 mM ATP) after they have been adsorbed to the carbon film so that a steady state might be restored after any filaments were broken. New results have suggested that the process of fixation by staining with uranyl acetate has a time resolution of milliseconds (Zhao and Craig, 2003a,b). We thus expect that the specimen preparation and fixation procedures should preserve the different structural states at the two filament ends. "
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    ABSTRACT: Proteins in the ADF/cofilin (AC) family are essential for rapid rearrangements of cellular actin structures. They have been shown to be active in both the severing and depolymerization of actin filaments in vitro, but the detailed mechanism of action is not known. Under in vitro conditions, subunits in the actin filament can treadmill; with the hydrolysis of ATP driving the addition of subunits at one end of the filament and loss of subunits from the opposite end. We have used electron microscopy and image analysis to show that AC molecules effectively disrupt one of the longitudinal contacts between protomers within one helical strand of F-actin. We show that in the absence of any AC proteins, this same longitudinal contact between actin protomers is disrupted at the depolymerizing (pointed) end of actin filaments but is prominent at the polymerizing (barbed) end. We suggest that AC proteins use an intrinsic mechanism of F-actin's internal instability to depolymerize/sever actin filaments in the cell.
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