title: Generation of differentially modified microtubules using in vitro enzymatic approaches.
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.
authors.affiliation: Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
authors.forename: Annapurna
authors.initials: A
authors.lastname: Vemu
authors.affiliation: Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Garnham
authors.affiliation: Biochemistry Core, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA.
authors.forename: Duck-Yeon
authors.initials: DY
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Antonina
authors.initials: A
authors.lastname: Roll-Mecak
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000032:analysis / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine; D000123:Acetyltransferases; C049182:alpha-tubulin acetylase; D010453:Peptide Synthases; C112698:tubulin polyglutamylase; C023213:tyrosyltubulin ligase
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)