Stepwise dynamics of connecting filaments measured in single myofibrillar sarcomeres

Single relaxed myofibrils of bumblebee flight muscle were subjected to motor-imposed ramp-length changes. The image of the striations was projected onto a linear photodiode array, and sarcomere length was computed as the spacing between centroids of contiguous A-bands. Centroid position was determined by integrating the respective A-band intensity peak and computing the location at which the area on one side was equal to the other. The resulting trace of centroid to centroid span versus time was stepwise, with periods of rapid shortening alternating with periods of pause. An alternative nondiscrete sensor gave similar steps. If thick filament length remains constant, stepwise sarcomere length changes imply that length changes in the connecting filament must be stepwise. Thus, shortening of the connecting filament occurs as a sequence of discrete events rather than as a continuous event.