Tropomyosins differentially impact the mechanical behaviour of cells

The influence of the actin cytoskeleton on the shape and mechanical properties of cells has been revealed extensively using anti-actin drugs. However, due to the lack of the specificity of these compounds for different actin filament populations it has been difficult to delineate the exact role of the actin cytoskeleton in these cellular features. Actin binding proteins largely affect the organisation and dynamics of actin in cells; however, the impact of these proteins on the mechanical properties of cells is still unknown. Tropomyosins (Tpms) differentially regulate actin filament stability, function and organisation through their highly regulated expression and sorting to specific intracellular sites. Hence, Tpm isoforms can be used as tools to identify and manipulate functionally distinct actin filament populations in a cell. In this study we investigated the impact of Tpm-containing actin filaments on the organisation of the actin cytoskeleton and the mechanical properties of cells using rat neuroblastoma cells stably overexpressing different Tpm isoforms in comparison with control cells. The mechanical measurements conducted by AFM showed that Tpm1.12-, Tpm4.2- and Tpm3.1-overexpressing cells were significantly stiffer than control cells. Measuring the total amount of Tpm expression in the Tpm-overexpressing cells demonstrated that there is no direct correlation between the modulus of Tpm-overexpressing cells and the total amount of Tpm expression. Biochemical evaluation of the filamentous to globular actin ratios demonstrated that the filamentous to globular actin ratio is higher in Tpm3.1 (M and H) cells than the controls. Also, computational analysis of the number of actin cables per cell revealed significant differences in the number of actin cables per cell relative to that seen in the control cells. The siRNA knockdown of Tpm3.1 in Tpm3.1-overexpressing cells caused the elastic modulus to revert to that of control cells. In addition, the inhibition of non-muscle myosin II in stiffer cells led to the significantly softer cells. The results show that the overexpression of Tpm changes the properties of the actin cables in an isoform-specific manner that is not directly related to cell stiffness. This suggests that it is specific aspects of actin organisation which regulate the mechanical properties of the cel