γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

The augmin complex architecture reveals structural insights into microtubule branching.

In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria.

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer-monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

Modular assembly of the principal microtubule nucleator γ-TuRC.

The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. Recent cryo-EM reconstructions visualized the intricate quaternary structure of the γ-TuRC, containing more than thirty subunits, raising fundamental questions about γ-TuRC assembly and the role of actin as an integral part of the complex. Here, we reveal the structural mechanism underlying modular γ-TuRC assembly and identify a functional role of actin in microtubule nucleation. During γ-TuRC assembly, a GCP6-stabilized core comprising GCP2-3-4-5-4-6 is expanded by stepwise recruitment, selective stabilization and conformational locking of four pre-formed GCP2-GCP3 units. Formation of the lumenal bridge specifies incorporation of the terminal GCP2-GCP3 unit and thereby leads to closure of the γ-TuRC ring in a left-handed spiral configuration. Actin incorporation into the complex is not relevant for γ-TuRC assembly and structural integrity, but determines γ-TuRC geometry and is required for efficient microtubule nucleation and mitotic chromosome alignment in vivo.

The gamma-tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator.

Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αß-tubulin. In higher eukaryotes, de novo assembly of microtubules from αß-tubulin is initiated by a 2 MDa multi-subunit complex, the gamma-tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer.

Reconstitution of the recombinant human γ-tubulin ring complex.

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

Microtubule nucleation: The waltz between γ-tubulin ring complex and associated proteins.

Microtubules are essential cytoskeletal elements assembled from αß-tubulin dimers. In high eukaryotes, microtubule nucleation, the de novo assembly of a microtubule from its minus end, is initiated by the γ-tubulin ring complex (γ-TuRC). Despite many years of research, the structural and mechanistic principles of the microtubule nucleation machinery remained poorly understood. Only recently, cryoelectron microscopy studies uncovered the molecular organization and potential activation mechanisms of γ-TuRC. In vitro assays further deciphered the spatial and temporal cooperation between γ-TuRC and additional factors, for example, the augmin complex, the phase separation protein TPX2, and the microtubule polymerase XMAP215. These breakthroughs deepen our understanding of microtubule nucleation mechanisms and will link the assembly of individual microtubules to the organization of cellular microtubule networks.

The structure of the γ-TuRC: a 25-years-old molecular puzzle.

The nucleation of microtubules from αß-tubulin dimers is an essential cellular process dependent on γ-tubulin complexes. Mechanistic understanding of the nucleation reaction was hampered by the lack of γ-tubulin complex structures at sufficiently high resolution. The recent technical developments in cryo-electron microscopy have allowed resolving the vertebrate γ-tubulin ring complex (γ-TuRC) structure at near-atomic resolution. These studies clarified the arrangement and stoichiometry of gamma-tubulin complex proteins in the γ-TuRC, characterized the surprisingly versatile integration of the small proteins MZT1/2 into the complex, and identified actin as an integral component of the γ-TuRC. In this review, we summarize the structural insights into the molecular architecture, the assembly pathway, and the regulation of the microtubule nucleation reaction.

The cryo-EM structure of a γ-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems.

The nucleation of microtubules from αß-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.

Insights into the assembly and activation of the microtubule nucleator γ-TuRC.

Microtubules are dynamic polymers of α- and ß-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αß-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction2. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors3. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis4.

Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau.

Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. We determined the binding sites of unphosphorylated and phosphorylated MAP2c responsible for interactions with the regulatory protein 14-3-3ζ. Differences in phosphorylation and in charge distribution between MAP2c and Tau suggested that both MAP2c and Tau respond to the same signal (phosphorylation by PKA) but have different downstream effects, indicating a signaling branch point for controlling microtubule stability. Although the interactions of phosphorylated Tau with 14-3-3ζ are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3ζ to MAP2c enhanced by PKA-mediated phosphorylation is likely to influence microtubule-MAP2c binding much less, in agreement with the results of our tubulin co-sedimentation measurements. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c also may regulate processes other than microtubule dynamics.

Phosphorylation of the regulatory domain of human tyrosine hydroxylase 1 monitored using non-uniformly sampled NMR.

Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its regulatory domain (RD-hTH1) and by an interaction with the 14-3-3 protein. The RD-hTH1 is composed of a structured region (66-169) preceded by an intrinsically disordered protein region (IDP, hTH1_65) containing two phosphorylation sites (S19 and S40) which are highly relevant for its increase in activity. The NMR signals of the IDP region in the non-phosphorylated, singly phosphorylated (pS40) and doubly phosphorylated states (pS19_pS40) were assigned by non-uniformly sampled spectra with increased dimensionality (5D). The structural changes induced by phosphorylation were analyzed by means of secondary structure propensities. The phosphorylation kinetics of the S40 and S19 by kinases PKA and PRAK respectively were monitored by non-uniformly sampled time-resolved NMR spectroscopy followed by their quantitative analysis.