Cytoskeletal Tension differences between Normal and Dystrophic Myotubes Probed with FRET Based Stress Sensors

Abstract

Duchene muscular dystrophy is caused by the loss of the cortical cytoskeletal protein dystrophin. These muscle cells have abnormally structured cortical cytoskeleton that leads to impaired ability to control stress in the cell cortex. When a stress bearing cytoskeletal element is removed from a system other proteins rearrange to adapt to the changed stress distribution and must absorb stresses that they were not intended to bear. This could affect a number of downstream mechanically sensitive receptors and enzymes. Knowing which cytoskeletal elements absorb the stress in the system that was intended for dystophin would be useful in understanding what mechanically based sensory systems will be affected most and can help in the design of treatments and in assessing therapies to treat muscular dystrophy. We created chimeric cytoskeletal proteins containing the cpstFRET stress sensing cassette and expressed them in developing normal and dystrophic mouse myotubes. These proteins included actinin, filamin, spectrin, vinculin and dystrophin. These chimeric proteins all showed distinct spatial distributions in the myotubes. We measured the stress on these proteins in resting cells and in cells stretched with a micropipette. All proteins had different resting stress levels. Filamin, an important component of focal adhesion plaques, showed the most significant difference in resting stress levels between normal and dystrophic myotubes. It also showed the most significant stress change in response to stretching. The distribution of filamin was very similar to dystrophin forming longitudinal tracks along the contacted surfaces of the myotube.