Reconstituting Dynamic Microtubule Polymerization Regulation by TOG Domain Proteins

Microtubules (MTs) polymerize from soluble alpha beta-tubulin and undergo rapid dynamic transitions to depolymerization at their ends. Microtubule-associated regulator proteins modulate polymerization dynamics in vivo by altering microtubule plus end conformations or influencing alpha beta-tubulin incorporation rates. Biochemical reconstitution of dynamic MT polymerization can be visualized with total internal reflection fluorescence (TIRF) microscopy using purified MT regulators. This approach has provided extensive details on the regulation of microtubule dynamics. Here, I describe a general approach to reconstitute MT dynamic polymerization with TOG domain microtubule regulators from the XMAP215/Dis1 and CLASP families using TIRE microscopy. TIRE imaging strategies require nucleation of microtubule polymerization from surface-attached, stabilized MTs. The approaches described here can be used to study the mechanism of a wide variety of microtubule regulatory proteins.