Nonequilibrium Bending Fluctuations Reveal the Mechanics of Microtubules and Mitochondria in Living Cells

Living systems operate far from thermodynamic equilibrium. Movements in cells are always driven by thermal forces but, more importantly, also by motor-generated active forces in the cytoplasm. How intracellular structures respond to these forces depends on their mechanical properties. Intracellular dynamics remain challenging to understand because, in most cases, neither local forces nor local material response properties are known, and imaging of fluctuations is typically not sufficient to measure both. While some subcellular structures such as actin filaments and microtubules, which form the major cytoskeletal networks, have been extensively studied , their mechanical properties in living cells where they are regulated by post-translational modifications and association with regulatory factors remain poorly understood. Here we present an alternative approach that uses nonequilibrium bending fluctuations of rod-shaped structures to characterize their mechanical properties and those of their surroundings and to estimate driving forces. We demonstrate that, when one of the three factors—two material properties and one driving force—is determined independently, the other two can be derived from observed fluctuations. By applying our method to microtubules in living cells we find that polyglutamylation, a post-translational modification enriched on microtubules that need to withstand large deformation forces like those in axons or cilia, increases microtubule stiffness. In contrast, tubelike mitochondria, lipid membrane-bounded organelles which easily deform, yielded a bending stiffness significantly lower than that of microtubules. Our method opens the door to a quantitative understanding of the effects of cellular factors on the mechanical properties of filaments and organelles in cells.