FIGURE 2.

Step-by-step motion of the isometric clamp. The sequential mechanism of the isometric force clamp with and without an actomyosin attachment can be summarized as follows. Without an actomyosin attachment, as in panel a, the clamp is stable so that the average laser forces on the two beads are equal and opposite. Even without an actomyosin attachment, the clamp responds to Brownian motions by reducing the fluctuations of the transducer bead while increasing the fluctuations of the motor bead. When myosin attaches and undergoes a power stroke, as in panel b, the actin and both beads immediately move toward the pointed end, decreasing the tension between the myosin and the transducer bead and increasing it between the myosin and the motor bead. As an example, the force produced by the myosin is shown as 2 pN in panel b. The amplified integral of the error signal from the transducer detector moves the motor trap to the right, further increasing the tension on the motor bead such that the transducer bead is returned toward its preattachment position, as shown in panel c. When the feedback loop settles, the transducer bead is returned to its preattachment position returning the actin, and therefore the myosin too, to its prepower stroke position. The measurement of the motor bead force (15 pN) is therefore equal to the exact force exerted by the myosin (10 pN) plus the pre-tension (5 pN), which can be subtracted from the measurement since this offset applied to the actin filament is known from the calibration process. Returning the actin to its preattachment position restores the myosin to its isometric condition while effectively eliminating the end compliance at the transducer bead/actin interface. When the myosin detaches, the actin and both beads move rapidly to the right. The amplified negative error signal from the transducer causes the motor trap to move left, decreasing the force on the motor bead and returning it to the state shown in panel a. The darker circles show the current bead position, and the lighter circles with a dashed circumference show the previous bead positions.