Reconstituting dynamic microtubule polymerization regulation by TOG domain proteins

Methods Enzymol. 2014:540:131-48. doi: 10.1016/B978-0-12-397924-7.00008-X.

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.

Keywords: CLASP; Microtubule dynamics; TIRF microscopy; TOG domains; Tubulin dimer; XMAP215/Dis1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Equipment Design
  • Fluorescent Dyes / analysis
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods*
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / metabolism
  • Microtubule-Associated Proteins / ultrastructure
  • Microtubules / chemistry
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Polymerization
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Tubulin / chemistry
  • Tubulin / metabolism*
  • Tubulin / ultrastructure

Substances

  • Fluorescent Dyes
  • Microtubule-Associated Proteins
  • Recombinant Proteins
  • Tubulin