Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

Electro-optical property of extremely stretched skinned muscle fibers.

Skinned fibers of frog semitendinosus muscle could easily be stretched up to 8 mum or more in sarcomere length. Such extremely stretched fibers gave quite sharp optical diffraction patterns. The intensities of all observable diffraction lines were found to increase on application of electric field (10 similar to 100 V/cm) parallel to the fiber axis, provided that there was no overlap between thin and thick filaments. By use of a polarizing microscope, it was concluded that I-bands were mainly responsible for this intensity increase. By application of square pulses, the time course of the intensity increase and decay was followed. The analysis based on a simple model suggests: (a) Each thin filament has a permanent dipole movement and the movement directs from Z-bands to the free end of the thin filament. (b) The flexural rigidity of thin filaments is estimated to be similar to 3 with 10-17 dyn with cm-2. The present fibers will provide various applications in physiochemical studies of in vivo thin and thick filaments