Muscles are crucial effectors of animal behaviour, as they translate neuronal signals into movement. Generally, most of the muscles are composed of structural and functional units called sarcomeres. Sarcomeres are pseudo-crystalline structures that are defined as the region between two Z-discs, at which parallel actin filaments are crosslinked. These actin filaments point to and overlap with the centrally located myosin filaments. The region surrounding the Z-discs, that is devoid of myosin is called the I-band, while the myosin containing region is called the A-band. During sarcomere contraction the I-band shrinks, while the A-band stays constant. The elasticity of the sarcomere is attributed to a long protein that spans the half-sarcomere, called titin. The I-band region of titin contains elastic Ig domains and PEVK regions, that largely contribute to the elasticity and bear the passive tension of the sarcomere.
The Drosophila I-band titin homolog is Sallimus (Sls), which spans from the Z-disc to the beginning of the myosin filament. Inserting an extra PEVK region into Sallimus (Sls) increases the length of the I-band and the Sls protein itself, as expected. Interestingly, it also results in an increase in actin and myosin filament lengths, resulting in a constant I-band to A-band ratio. This suggested a biomechanical feedback loop between actin and myosin filaments with the elastic titin, allowing for the scaling of sarcomere dimensions. However, the exact mechanism of this feedback on sarcomere mechanics remains to be explored.
During my PhD thesis, we aimed to investigate how the Sls PEVK region mediates biomechanical feedback between the components of the Drosophila sarcomere and test if changing the PEVK length changes the mechanical properties of the sarcomere. In aim 1, we mapped the viscoelastic properties of the different sarcomeric regions at high resolution in wild-type sarcomeres from adult flight and larval body wall muscles, using atomic force microscopy (AFM), and compared them with sarcomeres containing an extra-long PEVK Sls version. Our results show that in flight muscle sarcomeres, insertion of PEVK into Sls makes the I-band, and interestingly also the A-band, softer. We also see a specific increase in fluidity in the I-band. This suggests that the energy dissipation is higher, indicating inefficient muscles. The viscoelastic properties of the larval muscle sarcomeres show that they are an order of magnitude softer and more fluid-like than the flight muscle sarcomeres and, again insertion of extra PEVK into Sls further decreases their stiffness.
In the complementary aim 2, we have established a Drosophila larval stretching apparatus, which can stretch living wild-type or mutant larvae under the confocal microscope, while the sarcomere length can be monitored. This allowed to increase sarcomere length in a controlled way and image the dynamics of the sarcomere proteins. We are currently quantifying the force-length relationship in these larvae by mechanically stretching the muscles and inducing their contraction using optogenetics.