| Popis: |
Smooth muscle has the unique property of maintaining tension with low ATP consumption. It is generally accepted that this property, called the latch-state, results from the dephosphorylation of myosin while attached to actin. However, detached dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the effect of caldesmon on the binding force of unphosphorylated myosin to actin. We quantified the average unbinding force (Funb) in the absence or presence of caldesmon, ERK phosphorylated caldesmon, or caldesmon plus tropomyosin. Briefly, a microsphere captured in a single beam laser trap was attached to a fluorescently labeled actin filament that was then brought in contact with a pedestal coated with unphosphorylated myosin. The pedestal was then moved away from the trap at constant velocity. The actin/microsphere followed the pedestal until the force exerted by the trap on the microsphere exceeded the binding force of the unphosphorylated myosin to the actin. At this point, the microsphere sprang back into the trap center. Funb was calculated as the product of the trap stiffness and the maximal displacement of the microsphere from the trap center. Funb was normalized to the number of myosin molecules estimated per actin filament length. Funb from unregulated actin (0.09±0.01 pN) was significantly increased in the presence of caldesmon (0.17±0.02 pN), tropomyosin (0.17±0.02 pN) or both regulatory proteins (0.18±0.02 pN). Interestingly, ERK phosphorylation of caldesmon significantly reduced the Funb (0.06±0.01 pN). Thus, caldesmon enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. Conversely, ERK phosphorylation of caldesmon decreases this binding force to very low levels, suggesting a mechanism for muscle relaxation from the latch-state.Supported by: NSERC, NIH-RO1HL103405 |
