Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain.

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45 degrees line patterns subjected to CTS, the direction of maximum shear strain.

Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding.

Mechanical forces play a crucial role in controlling the integrity and functionality of cells and tissues. External forces are sensed by cells and translated into signals that induce various responses. To increase the detailed understanding of these processes, we investigated cell migration and dynamic cellular reorganisation of focal adhesions and cytoskeleton upon application of cyclic stretching forces. Of particular interest was the role of microtubules and GTPase activation in the course of mechanotransduction. We showed that focal adhesions and the actin cytoskeleton undergo dramatic reorganisation perpendicular to the direction of stretching forces even without microtubules. Rather, we found that microtubule orientation is controlled by the actin cytoskeleton. Using biochemical assays and fluorescence resonance energy transfer (FRET) measurements, we revealed that Rac1 and Cdc42 activities did not change upon stretching, whereas overall RhoA activity increased dramatically, but independently of intact microtubules. In conclusion, we demonstrated that key players in force-induced cellular reorganisation are focal-adhesion sliding, RhoA activation and the actomyosin machinery. In contrast to the importance of microtubules in migration, the force-induced cellular reorganisation, including focal-adhesion sliding, is independent of a dynamic microtubule network. Consequently, the elementary molecular mechanism of cellular reorganisation during migration is different to the one in force-induced cell reorganisation.