Ring Complex Research Articles

Molecular modeling reveals binding interface of γ-tubulin with GCP4 and interactions with noscapinoids.

The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/β-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface.

The Kivalliq Igneous Suite: Anorogenic bimodal magmatism at 1.75 Ga in the western Churchill Province, Canada

The Kivalliq Igneous Suite (1.77–1.73Ga) includes the Nueltin Granite intrusions; gabbro and anorthosite intrusions; three swarms of mafic dykes; and basalt, rhyolite and pyroclastic rocks of the Pitz Formation (Wharton Group, middle Dubawnt Supergroup). It was emplaced in the reworked Archaean hinterland of the trans-Hudson orogen, in the central western Churchill Province (WCP) of the Canadian Shield. The Nueltin granites have field relations, mineral assemblages, and compositions typical of metaluminous to peraluminous, Proterozoic to Recent rapakivi granites, and the suite comprises a classic bimodal anorogenic province. Kivalliq rhyolites/granites were erupted/emplaced in a N–S band (the “Nueltin corridor”) about 700km long and 300km wide, with volcanic rocks and dykes concentrated at the north end and coarse plutonic rocks to the south. The McRae Lake dyke swarm originates among three prominent gabbro/anorthosite intrusions inside the corridor, and forms a narrow array extending a minimum of 600km to the NE along the central axis of the WCP. Early mafic rocks (intrusions and some lavas, 1.77Ga) had an alkali basalt parent, with subalkaline basalts (sills, dykes, and mafic enclaves) present at the peak time of granite emplacement (1.75Ga). Average initial ɛNd of basaltic rocks at 1.75Ga is −4.0, indicating a significant crustal component. Sm–Nd depleted mantle model ages of granites and rhyolites (average initial ɛNd=−8.7) range from 2.2 to 3Ga, consistent with melted Archaean crust variably mixed with juvenile basalt. Metallogenic prospects of the Kivalliq Suite include U–Th–Zr–Y–REE enriched granite and syenite, epithermal Ag/Au at silicic centres (including mafic/felsic ring complexes), and Pb–Cu sulphides associated with mafic intrusions. Topaz rhyolites are present, but Sn-enriched rocks have not been observed. The Kivalliq Suite is one of about 15 prominent anorogenic provinces and dyke swarms which erupted within the Nuna supercontinent at ca. 1.75Ga, and the thermal effects of the Kivalliq event are recorded in U–Pb and K–Ar data throughout most of the western Churchill Province.

Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.

Early Cretaceous counterclockwise rotation of Northeast Africa within the equatorial zone: Paleomagnetic study on Mansouri ring complex, Southeastern Desert, Egypt

The Mansouri ring complex (132Ma) is, paleomagnetically, studied to shed light on the paleo-tectonic position of Northeast Africa during the Early Cretaceous. Progressive thermal demagnetization of all samples verified a general bi-vectorial decay of the natural remanence. After the removal of the present-day field overprint, the decaying anchored component was either:1.A dual-polarity, shallow NW–SE directed component residing in magnetite (400–585°C) of shiny fresh samples, or,2.A normal-polarity, medium-inclination, north-oriented component stored in haematite of few reddish ferruginous sites. This component was considered as chemical remagnetization carried by secondary haematite.Due to its steady stability, overwhelming existence in most sites, positive reversal test and its residence in fresh-samples’ magnetite, the first dual-polarity, shallow NW–SE component, was considered as the characteristic remanent magnetization [ChRM] representing the paleomagnetic field during cooling of the Mansouri ring complex. The mean paleomagnetic pole of the isolated ChRM was at 47°N/259°E, Dp/Dm=3.4°/6.6°.This Hauterivian pole from Egypt shows reasonable consistency with its coeval poles rotated from the main tectonic units to Northeast Africa. It reveals that in Early Cretaceous:1.Northeast Africa was equatorial, lying just south the Equator. Cairo, which is now at 30°N, was at −1.5° paleo-latitude.2.The Azimuth of the African Plate was NE–SW, about 30° clockwise with respect to the present-dayN–S trend.Comparing this Hauterivian pole to that of the Wadi Natash basalts [107±4Ma], which was at [55°N/250°E] during the Albian, the African Plate seems to have rotated counter-clockwise about 10° with Northeast Africa moving northwards [Cairo was moving from 1.5°S to 1.5°N] within the equatorial zone, during the Early Cretaceous.

Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.

Mitochondrial dysfunction and alterations in energy metabolism have been implicated in a variety of human diseases. Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Here, we provide a link between PHB2 deficiency and hyperactive insulin/IGF-1 signaling. Deletion of PHB2 in podocytes of mice, terminally differentiated cells at the kidney filtration barrier, caused progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF-1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF-1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2-deficient animals. Evidently, perturbation of insulin/IGF-1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

Coordination complexes of thiazyl rings — Synthesis, structure, and density functional theory (DFT) computational analysis of CpCr(CO)x (x = 2, 3) complexes of fluorinated and nonfluorinated 1λ3-1,2,4,6-thiatriazinyls with differing Cr–S bond orders

The reaction of [3,5-Ph2-C2N3S]2 with [CpCr(CO)3]2 in toluene at room temperature forms an adduct via a Cr–S bond, formulated as CpCr(CO)3SN3C2Ph2, which has fitting NMR, IR, and combustion analysis data. The structure was determined by a single-crystal X-ray structure diffraction study (P21/n, a = 8.4611(17) Å, b = 20.509(4) Å, c = 11.757(2) Å, β = 104.453(7)°). The Cr–S bond length of 2.4908(11) Å corresponds to a bond order of 1.0 from >90 values for CpCr(CO)x or Cp*Cr(CO)x moieties (x = 2, 3) bonded to S, which are used to establish a Pauling-type bond order scale specific to this class of compounds. Similar reactions of fluorinated thiatriazinyls derived from [3-Ph-5-CF3-C2N3S]2 or [4-MeOC6H4-5-CF3-C2N3S]2 are accompanied by the loss of CO to produce CpCr(CO)2SN3C2PhCF3 (P1, a = 8.0929(8) Å, b = 10.3160(10) Å, c = 11.2405(11) Å, α = 70.032(2)°, β = 72.076(2)°, γ = 82.375(2)°) and CpCr(CO)2SN3(CCF3)(C6H4OCH3) (P21/c, a = 8.1311(7) Å, b = 24.284(2) Å, c = 9.1025(8) Å, β = 97.218(2)°), also fully characterized by spectroscopy and crystallography. Their measured Cr–S bond lengths, 2.2987(14) and 2.2965(11) Å, correspond to bond orders of 1.5. (U/R)B3PW91/6-311+G(2df,2p)//B3PW91/6-31G(2d,p) hybrid density functional theory (DFT) calculations show that the tricarbonyl complex has an unusual σ bond. However, the dicarbonyl complexes of the fluorinated thiatriazinyls are π bonded.

The kinesin–tubulin complex: considerations in structural and functional complexity

The kinesin–tubulin complex: considerations in structural and functional complexity Zachary T Olmsted, Andrew G Colliver, Janet L Paluh State University of New York Polytechnic Institute, Colleges of Nanoscale Science and Engineering, College of Nanoscale Science, Nanobioscience Constellation, Albany, NY, USA Abstract: The ability of cells to respond to external cues by appropriately manipulating their internal environment requires a dynamic microtubule cytoskeleton that is facilitated by associated kinesin motor interactions. The evolutionary adaptations of kinesins and tubulins when merged generate a highly adaptable communication and infrastructure cellular network that is important to understanding specialized cell functions, human disease, and disease therapies. Here, we review the state of the field in the complex relationship of kinesin–tubulin interactions. We propose 12 mechanistic specializations of kinesins. In one category, referred to as sortability, we describe how kinesin interactions with tubulin isoforms, isotypes, or posttranslationally modified tubulins contribute to diverse cellular roles. Fourteen kinesin families have previously been described. Here, we illustrate the great depth of functional complexity that is possible in members within a single kinesin family by mechanistic specialization through discussion of the well-studied Kinesin-14 family. This includes new roles of Kinesin-14 in regulating supramolecular structures such as the microtubule-organizing center γ-tubulin ring complex of centrosomes. We next explore the value of an improved mechanistic understanding of kinesin–tubulin interactions in regard to human development, disease mechanisms, and improving treatments that target kinesin–tubulin complexes. The ability to combine the current kinesin nomenclature along with a more precisely defined kinesin and tubulin molecular toolbox is needed to support more detailed exploration of kinesin–tubulin interaction mechanisms including functional uniqueness, redundancy, or adaptations to new roles upon cell specialization, and to thereby accelerate applications in human health. Keywords: microtubule, cellular network, cell functions, sortability, transport, disease, signaling

Ring closure activates yeast γTuRC for species-specific microtubule nucleation.

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm its functional importance we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, suggesting this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a profound preference for tubulin from the same species.

Suppression of centrosome protein TACC3 induces G1 arrest and cell death through activation of p38–p53–p21 stress signaling pathway

The centrosome regulates diverse cellular processes, including cell proliferation and differentiation. TACC3, a member of the human transforming acidic coiled-coil protein family, is a key centrosomal protein that is up-regulated in many cancers. Previous studies have demonstrated that TACC3 is essential for the survival of vertebrates and is involved in cell cycle regulation in human cells. However, the details of the underlying mechanisms in its cell cycle regulatory activity remain poorly understood. In this study, we showed that suppression of TACC3 expression induced G1 cell cycle arrest and triggered cell death in human cells. TACC3 depletion-induced G1 arrest and cell death were significantly reduced in cells either lacking p53 or with pharmacologically-inhibited p38, indicating that G1 arrest and cell death induction both require p53 and p38. TACC3 depletion up-regulated the levels of p53 and p21 and induced the accumulation of p53 both in the nucleus and at the centrosome. Interestingly, TACC3 depletion led to the activation of p38 and stimulated the recruitment of activated p38 to the centrosome. Depletion of TACC3 up-regulated the phosphorylation of p53 at Serine 33, a site known to be phosphorylated by p38 under cellular stress and further induced the accumulation of phosphorylated p53 to the centrosome. Loss of TACC3 affected centrosome integrity by disrupting the localization of components of the γ-tubulin ring complex at the centrosome. The results demonstrate that TACC3 depletion induces G1 arrest and cell death by activating p38–p53–p21 signaling and triggering a centrosome-mediated cellular stress response.

Structure and function of the bacterial flagellar type III protein export system in Salmonella

The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

ChemInform Abstract: Antimicrobial Activity of Quinoxaline Derivatives

AbstractReview: 50 refs.

Dualizing complexes of seminormal affine semigroup rings and toric face rings

We characterize the seminormality of an affine semigroup ring in terms of the dualizing complex, and the normality of a Cohen–Macaulay semigroup ring by the “shape” of the canonical module. We also characterize the seminormality of a toric face ring in terms of the dualizing complex. A toric face ring is a simultaneous generalization of Stanley–Reisner rings and affine semigroups.

Targeting of γ-tubulin complexes to microtubule organizing centers: conservation and divergence

Organisms with closed or open mitosis have differentially evolved various gamma-tubulin complex (γ-TuC) recruiting factors to organize diverse cellular microtubule (MT) arrays, including the mitotic spindle. γ-TuC recruiting factors not only target the γ-TuC to MT nucleation sites, but also regulate MT nucleation activity by generating the template for MT nucleation or promoting the MT nucleation activity of pre-existing γ-tubulin ring complexes (γ-TuRCs). Here we outline the current understanding of MT nucleator assembly and its regulation by γ-tubulin small complex (γ-TuSC) receptors. Moreover, we discuss the emergence of γ-TuC recruiting factors through evolution with augmented complexity and diversity and propose a hypothesis to account for the evolution of these factors in cooperative spindle assembly.

Microtubule Nucleation in Mitosis by a RanGTP-Dependent Protein Complex

The γ-tubulin ring complex (γTuRC) is a multisubunit complex responsible for microtubule (MT) nucleation in eukaryotic cells. During mitosis, its spatial and temporal regulation promotes MT nucleation through different pathways. One of them is triggered around the chromosomes by RanGTP. Chromosomal MTs are essential for functional spindle assembly, but the mechanism by which RanGTP activates MT nucleation has not yet been resolved. We used a combination of Xenopus egg extracts and in vitro experiments to dissect the mechanism by which RanGTP triggers MT nucleation. In egg extracts, NEDD1-coated beads promote MT nucleation only in the presence of RanGTP. We show that RanGTP promotes a direct interaction between one of its targets, TPX2, and XRHAMM that defines a specific γTuRC subcomplex. Through depletion/add-back experiments using mutant forms of TPX2 and NEDD1, we show that the activation of MT nucleation by RanGTP requires both NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex through a TPX2-dependent mechanism. The XRHAMM-γTuRC complex is the target for activation by RanGTP that promotes an interaction between TPX2 and XRHAMM. The resulting TPX2-RHAMM-γTuRC supracomplex fulfills the two essential requirements for the activation of MT nucleation by RanGTP: NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex onto a TPX2-dependent scaffold. Our data identify TPX2 as the only direct RanGTP target and NEDD1 as the only Aurora A substrate essential for the activation of the RanGTP-dependent MT nucleation pathway.

Physicochemical Evolution of the Thermal Springs over the Siwana Ring Complex, Western Rajasthan

Abstract: The chemical composition of thermal springs from Siwana Ring Complex (SRC) of Barmer district, Rajasthan, India has been investigated for the first time. These springs are near neutral to mildly alkaline (pH = 6.8 to 7.8) in nature with surface temperatures varying between 31 to 39 °C. Piper diagram suggests that these thermal springs are dominated by Ca-HCO3 type. Experimental results of water-rock interaction at 100 °C indicate that the thermal springs are circulating through tuff and a sedimentary formation extensively controlled by ring dykes of granites, felsic volcanics and mafic dyke and the fault systems associated with the host rock. Groundwater and thermal springs show similar characteristics. Estimated reservoir temperature suggests that Siwana area geothermal system is a low enthalpy system. Heat flow values of the area range from 83 to 205 mWm−2, promise a viable potential for Enhanced Geothermal System (EGS).

Deubiquitination of γ-tubulin by BAP1 prevents chromosome instability in breast cancer cells.

Microtubule nucleation requires the γ-tubulin ring complex, and during the M-phase (mitosis) this complex accumulates at the centrosome to support mitotic spindle formation. The posttranslational modification of γ-tubulin through ubiquitination is vital for regulating microtubule nucleation and centrosome duplication. Blocking the BRCA1/BARD1-dependent ubiquitination of γ-tubulin causes centrosome amplification. In the current study, we identified BRCA1-associated protein-1 (BAP1) as a deubiquitination enzyme for γ-tubulin. BAP1 was downregulated in metastatic adenocarcinoma breast cell lines compared with noncancerous human breast epithelial cells. Furthermore, low expression of BAP1 was associated with reduced overall survival of patients with breast cancer. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Importantly, rescue experiments including expression of full length but not the catalytic mutant of BAP1 reduced ubiquitination of γ-tubulin and prevented mitotic defects. Our study uncovers a new mechanism for BAP1 involved in deubiquitination of γ-tubulin, which is required to prevent abnormal mitotic spindle formation and genome instability.

Single zircon Hf–O isotope constraints on the origin of A-type granites from the Jabal Al-Hassir ring complex, Saudi Arabia

The Jabal Al-Hassir ring complex in the southern Arabian Shield is an alkaline granite complex comprising an inner core of biotite granite that outwardly becomes a porphyritic sodic-calcic amphibole (ferrobarroisite–katophorite) granite. A combined study of mineral chemistry and single zircon Hf–O zircon isotope analyses was carried out to infer the magma sources of the Neoproterozoic post-collisional A-type granitoids in Saudi Arabia. The granitic rocks show high positive initial ɛHf(t) values of +7.0 to +10.3 and δ18O values of +5.8‰ to +7.4‰ that are consistent with melting of a juvenile crustal protolith that was formed during the Neoproterozoic assembly of the Arabian-Nubian Shield (ANS). Crustal-model ages (Hf-tNC) of 0.71–0.94Ga indicate minor contribution from an older continental crust in the formation of the Jabal Al-Hassir granitic rocks (crystallization age=620±3Ma), but any such component is likely to be Neoproterozoic in age. Temperature and oxygen fugacity (ƒO2) estimates suggested that the Jabal Al-Hassir A-type granite magma was generated at high temperature (820–1050°C) and low ƒO2. Geochemical characteristics (e.g., low ƒO2), geochronological data, and Hf and O isotope compositions, indicate that the magmas of the Neoproterozoic A-type granites of the Jabal Al-Hassir ring complex were likely generated by crustal partial melting of a juvenile Neoproterozoic lower crustal tholeiitic rocks, following collision between East and West Gondwana in the final stages of the evolution of the Arabian Shield.

TACC3 Protein Regulates Microtubule Nucleation by Affecting γ-Tubulin Ring Complexes

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.

Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the γ-tubulin ring complex (γ-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to γ-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at γ-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.

GCP-WD Mediates γ-TuRC Recruitment and the Geometry of Microtubule Nucleation in Interphase Arrays of Arabidopsis

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis, but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1, associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.