Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length.
Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length l(p). Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of l(p) on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of l(p) for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 mum, we find a systematic increase of l(p) from 110 to 5,035 mum. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature.