title: Reconstituting dynamic microtubule polymerization regulation by TOG domain proteins.
abstract: Microtubules (MTs) polymerize from soluble αβ-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 αβ-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 TIRF microscopy. TIRF 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.
authors.affiliation: Molecular Cellular Biology, University of California Davis, California, USA. Electronic address: jawdat@ucdavis.edu.
authors.forename: Jawdat
authors.initials: J
authors.lastname: Al-Bassam
mesh_terms: D000818:Animals; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D058105:Polymerization; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D005456:Fluorescent Dyes; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D014404:Tubulin
keywords: CLASP; Microtubule dynamics; TIRF microscopy; TOG domains; Tubulin dimer; XMAP215/Dis1