Abstract
For muscles to effectively power locomotion, trillions of myosin molecules must rapidly attach and detach from the actin thin filament. This is accomplished by precise regulation of the availability of the myosin binding sites on actin (i.e. activation). Both calcium (Ca++) and myosin binding contribute to activation, but both mechanisms are simultaneously active during contraction, making their relative contributions difficult to determine. Further complicating the process, myosin binding accelerates the attachment rate of neighboring myosin molecules, adding a cooperative element to the activation process. To de-convolve these two effects, we directly determined the effect of Ca++ on the rate of attachment of a single myosin molecule to a single regulated actin thin filament, and separately determined the distance over which myosin binding increases the attachment rate of neighboring molecules. Ca++ alone increases myosin’s attachment rate ~50-fold, while myosin binding accelerates attachment of neighboring molecules 400 nm along the actin thin filament.
Highlights
At the molecular level, vertebrate striated muscle contraction is regulated by the actin binding proteins troponin (Tn) and tropomyosin (Tm) in a calcium- (Ca++-) dependent process
Be answered: 1) how does Ca++ affect myosin binding to a regulated thin filaments (RTFs) at the single molecule level? and 2) how does Ca++ affect the coupling between nearby myosin molecules? To answer these questions, we performed measurements of myosin’s interaction with RTFs at a range of Ca++ concentrations using single molecule[30] and mini-ensemble (~14 independent myosin heads)[31, 32] laser trap assays, and using the in vitro motility assay with a large myosin ensemble (~75 independent myosin heads)
When we performed this assay with an actin filament including regulatory proteins (Fig. 1a), single myosin molecules bound to the RTF at a frequency of 0.85 ± 0.33 s−1 at saturating Ca++
Summary
Vertebrate striated muscle contraction is regulated by the actin binding proteins troponin (Tn) and tropomyosin (Tm) in a calcium- (Ca++-) dependent process. The molecular basis for this coupling is that, when myosin binds strongly to actin, it displaces Tm and locally activates the RTF9, 13, 20–23 In addition to this type of intermolecular coupling, myosin molecules interacting with a common RTF are mechanochemically coupled because forces generated by one motor affect the ADP release rate of other bound motors[24,25,26,27]. Both types of intermolecular coupling are forms of cooperativity, activation of a RTF by myosin strong binding acts locally (on the scale of ~100 nm28), while mechanochemical coupling acts more globally (on the scale of ~10 μm[27]). Since each of these experiments was performed at a different myosin surface density, the coupling between molecules was varied, from no coupling (single molecule laser trap), to weak coupling (small ensemble laser trap) to strong coupling (motility)
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