In vitro Microtubule Binding Assay and Dissociation Constant Estimation

Abstract

Microtubules (MTs) support an astonishing set of versatile cellular functions ranging from cell division, vesicle transport, and cell and tissue morphogenesis in various organisms. This versatility is in large mediated by MT-associated proteins (MAPs). The neuronal MAP Tau, for example, is stabilizing MTs in axons of the vertebrate nervous system and thus provides the basis for enduring axonal transport and the long life span of neurons (Mandelkow et al., 1994). Tau has been shown to bind to MTs directly in vitro and also to promote their nucleation from α-/β-tubulin subunits (Goode et al., 1994). Recently, we identified a plant-specific protein family called “companion of cellulose synthase” (CC), which was shown to bind MTs and enhance dynamics of the cortical MT array in plant cells under salt stress (Endler et al., 2015). The CCs were therefore hypothesized to help plant cells cope with stress conditions and thereby maintain biomass production under adverse growth conditions. Here, we provide detailed experimental information on in vitro MT binding assays, which allow assessing whether a protein of interest is binding to MTs. The assay utilizes the high molecular weight of MTs in a spin down approach and enables the determination of the dissociation constant Kd, a measure for the protein’s binding strength to MTs.