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Methods Enzymol. 2014;540:149-66. doi: 10.1016/B978-0-12-397924-7.00009-1.

Generation of differentially modified microtubules using in vitro enzymatic approaches.

Author information

1
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
2
Biochemistry Core, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA.
3
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA; Center for Biophysics, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA. Electronic address: antonina@mail.nih.gov.

Abstract

Tubulin, the building block of microtubules, is subject to chemically diverse and evolutionarily conserved post-translational modifications that mark microtubules for specific functions in the cell. Here we describe in vitro methods for generating homogenous acetylated, glutamylated, or tyrosinated tubulin and microtubules using recombinantly expressed and purified modification enzymes. The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the behavior of motors and microtubule-associated proteins.

KEYWORDS:

Microtubule; Motors; Tubulin acetylation; Tubulin acetyltransferase (α-TAT); Tubulin glutamylation; Tubulin post-translational modifications; Tubulin tyrosination; Tubulin tyrosine ligase (TTL); Tubulin tyrosine ligase-like (TTLL)

[Indexed for MEDLINE]

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