A Real-Space Method to Measure the Persistence Length of Dynamic Microtubules

Abstract

Microtubules play important roles in regulating the mechanical properties of the cytoskeleton filament system. While microtubules in cells often appear highly curved, microtubules grown in vitro tend to be straight. Therefore, a basic understanding of the mechanical properties of microtubules is essential to understand how they respond to extracellular forces and the binding of microtubule associated proteins and motors. The main techniques used to determine the persistence length of microtubules are either passive, relying on thermal fluctuations, or active, where external forces are applied via optical traps or fluid flow. Many of these studies used drug-stabilized microtubules that are not dynamic. However, stabilizing agents impact microtubule material properties. Existing methods to measure the persistence length of dynamic microtubules require long microtubules (over ∼30 um) and the use of non-intuitive ‘pseudo-modes’ to describe microtubule fluctuations. We present a simple real-space method to infer the persistence length of dynamic microtubules from their thermal fluctuations. We test this method on simulated dynamic microtubules and then apply it to determine the persistence length of dynamic, label-free microtubules imaged via darkfield microscopy. The combination of label-free imaging and a simple real-space analysis method is a powerful tool to investigate the mechanical properties of dynamic microtubules.