Chapter Three - Quantitative Analysis of Microtubule Self-assembly Kinetics and Tip Structure

Abstract

Microtubules are dynamic polymers of the cytoskeleton, which play important roles in cell division, polarization, and intracellular transport. Self-assembly of microtubule polymer from αβ-tubulin heterodimers is highly variable, with stochastic switching between alternate states of net growth and net shortening, a phenomenon known as dynamic instability. Microtubule tip structures are also variable and directly influence the kinetics of assembly and vice versa. TipTracker, a semiautomated, image processing-based tool, permits high spatial and temporal resolution measurements from fluorescence microscopy images (~10-40 nm, or 1-5 dimer lengths, at 1-10 Hz) with simultaneous tip structure estimation. We provide a walkthrough of the TipTracker code to demonstrate methods used to (1) fit the coordinates of the microtubule backbone; (2) track microtubule tip position; and (3) estimate tip structure from the spatial decay of the tip fluorescence distribution, discuss possible sources of error, and include an example protocol for nanometer-scale tip tracking in living cells. Additionally, we evaluate TipTracker's accuracy on simulated digital images and fixed microtubules to estimate accuracy under realistic imaging conditions. In summary, this chapter demonstrates the use of TipTracker in making robust, high-resolution measurements of microtubule tip dynamics and structures, facilitating quantitative investigations into nanoscale/molecular control of microtubule assembly. Although our primary focus is on microtubules, these methods are, in principle, suitable for other polymer structures, such as F-actin.