title: Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.
abstract: Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.
authors.affiliation: From the Cell Biology and Biophysics Unit, NINDS, and.
authors.forename: Annapurna
authors.initials: A
authors.lastname: Vemu
authors.affiliation: the Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London WC1E, United Kingdom.
authors.forename: Joseph
authors.initials: J
authors.lastname: Atherton
authors.affiliation: From the Cell Biology and Biophysics Unit, NINDS, and.
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Spector
authors.affiliation: From the Cell Biology and Biophysics Unit, NINDS, and.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Szyk
authors.affiliation: the Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London WC1E, United Kingdom c.moores@mail.cryst.bbk.ac.uk.
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Moores
authors.affiliation: From the Cell Biology and Biophysics Unit, NINDS, and Biophysics Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and Antonina@mail.nih.gov.
authors.forename: Antonina
authors.initials: A
authors.lastname: Roll-Mecak
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy; D006801:Humans; D008870:Microtubules; D008958:Models, Molecular; D009474:Neurons / Q000648:ultrastructure; D020033:Protein Isoforms / Q000737:chemistry / Q000648:ultrastructure; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D061987:Sf9 Cells; D018411:Spodoptera; D014404:Tubulin / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; C498585:TUBB3 protein, human; D014404:Tubulin
keywords: cryo-EM; cryo-electron microscopy; cytoskeleton; dynamic instability; engineered tubulin; microscopy; microtubule; microtubule dynamics; microtubule structure; post-translational modification; recombinant tubulin; tubulin; tubulin isoform