Ring Complex Research Articles

In vitro reconstitution of branching microtubule nucleation.

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed 'branching' MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

Biochemical reconstitution of branching microtubule nucleation.

Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways and their mode of action still need to be identified. Using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, which is critical for chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the branching effectors augmin and TPX2 are required to efficiently nucleate microtubules from pre-existing microtubules. TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice. TPX2 and augmin enable γ-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly-spaced patches along microtubules. This work provides a blueprint for other microtubule nucleation pathways and helps explain how microtubules are generated in the spindle.

Structure and tectonic setting of the Chingale Igneous Ring Complex, Malawi from aeromagnetic and satellite gravity data: Implication for Precambrian terranes collision and Neogene - Quaternary rifting

This work examines the structure and tectonic setting of the possibly late Neoproterozoic - early Cambrian Pan-African (henceforth Pan-African) Chingale Igneous Ring Complex (CIRC) in southern Malawi to document its internal emplacement structure and solicit implications for Precambrian terranes collision and Neogene – Quaternary rifting. The CIRC is located along the boundary between the Mesoproterozoic – Neoproterozoic Southern Irumide orogenic belt in the northwest (dominantly amphibolite facies metamorphic terrane) and the Unango complex to the southeast (dominantly granulite facies metamorphic terrane). Portion of the Southern Irumide orogenic belt has been recently recognized as the Niassa craton. The CIRC is also dissected by the southeastern border fault of the Malawi rift, which represents a segment of the Neogene – Quaternary Western Branch of the East African Rift System. This work uses reduced to the pole (RTP) aeromagnetic data enhanced through tilt derivative and three-dimensional (3D) magnetic susceptibility modeling to reveal that the CIRC comprises three overlapping 7 km–12 km wide circular to elliptical ring structures that are aligned in a NE-SW direction along the boundary between the Southern Irumide orogenic belt and the Unango complex. This work also uses Bouguer gravity anomalies of the World Gravity model 2012 (WGM 2012) subjected to upward continuation of 2 km and 12 km to show that the CIRC is located along a relatively steep gravity anomaly gradient coinciding with the boundary between the Southern Irumide orogenic belt and the Unango complex. Together with structural, lithological, metamorphic, and lithospheric structure observations, this work proposes that the CIRC mark the location of a Pan-African suture zone resulting from the collision of the Southern Irumide orogenic belt (Niassa craton) and the Unango complex and this is named here the Chingale suture zone. The presence of this Pan-African suture zone facilitated localization of extensional strain during the onset of the Malawi rift allowing it to change its orientation from NNW-trending in its northern part to NE-trending in its southern part. Igneous ring complexes are hypabyssal intrusions. Hence, the exposure of the CIRC within the southern Malawi rift suggests the lack of rift-related subsidence. This makes much of the ~300 m–~500 m high relief of the topographic escarpment of the southeastern border fault of the rift the result of rift flank uplift.

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

Microtubules are dynamic polymers of α- and β-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αβ-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 4.8Å 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.

Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex

The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at ∼3.8Å resolution, revealing anasymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000Å2) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Protein Misfolding Diseases and Therapeutic Approaches.

Protein folding is the process by which a polypeptide chain acquires its functional, native 3D structure. Protein misfolding, on the other hand, is a process in which protein fails to fold into its native functional conformation. This misfolding of proteins may lead to precipitation of a number of serious diseases such as Cystic Fibrosis (CF), Alzheimer's Disease (AD), Parkinson's Disease (PD), and Amyotrophic Lateral Sclerosis (ALS) etc. Protein Quality-control (PQC) systems, consisting of molecular chaperones, proteases and regulatory factors, help in protein folding and prevent its aggregation. At the same time, PQC systems also do sorting and removal of improperly folded polypeptides. Among the major types of PQC systems involved in protein homeostasis are cytosolic, Endoplasmic Reticulum (ER) and mitochondrial ones. The cytosol PQC system includes a large number of component chaperones, such as Nascent-polypeptide-associated Complex (NAC), Hsp40, Hsp70, prefoldin and T Complex Protein-1 (TCP-1) Ring Complex (TRiC). Protein misfolding diseases caused due to defective cytosolic PQC system include diseases involving keratin/collagen proteins, cardiomyopathies, phenylketonuria, PD and ALS. The components of PQC system of Endoplasmic Reticulum (ER) include Binding immunoglobulin Protein (BiP), Calnexin (CNX), Calreticulin (CRT), Glucose-regulated Protein GRP94, the thiol-disulphide oxidoreductases, Protein Disulphide Isomerase (PDI) and ERp57. ER-linked misfolding diseases include CF and Familial Neurohypophyseal Diabetes Insipidus (FNDI). The components of mitochondrial PQC system include mitochondrial chaperones such as the Hsp70, the Hsp60/Hsp10 and a set of proteases having AAA+ domains similar to the proteasome that are situated in the matrix or the inner membrane. Protein misfolding diseases caused due to defective mitochondrial PQC system include medium-chain acyl-CoA dehydrogenase (MCAD)/Short-chain Acyl-CoA Dehydrogenase (SCAD) deficiency diseases, hereditary spastic paraplegia. Among therapeutic approaches towards the treatment of various protein misfolding diseases, chaperones have been suggested as potential therapeutic molecules for target based treatment. Chaperones have been advantageous because of their efficient entry and distribution inside the cells, including specific cellular compartments, in therapeutic concentrations. Based on the chemical nature of the chaperones used for therapeutic purposes, molecular, chemical and pharmacological classes of chaperones have been discussed.

The spindle pole body of Aspergillus nidulans is asymmetrical and contains changing numbers of γ-tubulin complexes.

Centrosomes are important microtubule-organizing centers (MTOCs) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) are found in many cell types. Their composition and structure are only poorly understood. Here, we analyzed nuclear MTOCs (spindle-pole bodies, SPBs) and septal MTOCs in Aspergillus nidulans They both contain γ-tubulin along with members of the family of γ-tubulin complex proteins (GCPs). Our data suggest that SPBs consist of γ-tubulin small complexes (γ-TuSCs) at the outer plaque, and larger γ-tubulin ring complexes (γ-TuRC) at the inner plaque. We show that the MztA protein, an ortholog of the human MOZART protein (also known as MZT1), interacted with the inner plaque receptor PcpA (the homolog of fission yeast Pcp1) at SPBs, while no interaction nor colocalization was detected between MztA and the outer plaque receptor ApsB (fission yeast Mto1). Septal MTOCs consist of γ-TuRCs including MztA but are anchored through AspB and Spa18 (fission yeast Mto2). MztA is not essential for viability, although abnormal spindles were observed frequently in cells lacking MztA. Quantitative PALM imaging revealed unexpected dynamics of the protein composition of SPBs, with changing numbers of γ-tubulin complexes over time during interphase and constant numbers during mitosis.This article has an associated First Person interview with the first author of the paper.

Configurations of lead(II)–benzohydroxamic acid complexes in colloid and interface: A new perspective

Lead(II)–benzohydroxamic acid (Pb–BHA) complex collectors perform well with respect to scheelite flotation, and, due to their structure, they are widely used for industrial purposes. This paper examines the controversial issue of whether “O, O” five-membered ring or “N, O” four-membered ring complexes are formed when BHA coordinates with Pb ions, with their structure being comprehensively studied from the aspect of colloid and interface science. The configurations of Pb–BHA complexes are examined in a solution and on a mineral surface with experimental and computational methods. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) revealed that the five-membered ring is the dominant form of Pb–BHA complexes in a solution, whereas four-membered ring complexes are the stronger electron acceptor of the two. Moreover, XPS and time-of-flight secondary ion mass spectrometry (TOF-SIMS) confirmed that the four-membered ring complexes are stable with respect to being adsorbed on the scheelite surface. Therefore, although the four-membered ring is not as stable as the five-membered ring in a solution, it offers advantages with respect to adsorption on an electron-rich mineral surface during short-flotation processes.

The γ-tubulin complex protein GCP6 is crucial for spindle morphogenesis but not essential for microtubule reorganization in Arabidopsis

γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the GCP6 gene encoding the largest subunit of the γTuRC in Arabidopsis thaliana. Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. The gcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although the gcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. The gcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization.

Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing \u03b3-tubulin ring complex

BackgroundAstral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood.ResultsHere, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components.ConclusionsThe results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.

Synthesis and structural characterization of iridium(I) complexes of 20-membered macrocyclic rings bearing chelating bis(N-heterocyclic carbene) ligands.

The reactivities of two 20-membered macrocyclic ligands, each containing two N-heterocyclic carbene (NHC) and two amine groups, towards [IrCl(COD)]2 (COD is cycloocta-1,5-diene) were investigated. Macrocycles containing imidazolin-2-ylidene groups formed the monometallic complex [(1,2,5,6-η)-cycloocta-1,5-diene](5,16-dibenzyl-1,5,9,12,16,20-hexaazatricyclo[18.2.1.19,12]tetracosa-10,21-dien-21,22-diylidene)iridium(I) bromide dichloromethane monosolvate, [Ir(C8H12)(C32H42N6)]Br·CH2Cl2, 2a. The structure of iridium complex 2a at 100 K has triclinic P-1 symmetry. The ligand in 2a coordinates to the Ir center through the NHC moieties in a cis fashion. Additionally, the ligand adopts an umbrella-like structure that appears to envelope the Ir center. The structure displays C-H...Br interactions. Macrocycles containing benzimidazolin-2-ylidene groups formed the bimetallic complex [μ-5,20-dibenzyl-1,5,9,16,20,24-hexaazapentacyclo[22.6.1.19,16.010,15.025,30]dotriaconta-10(15),11,13,25(30),26,28-hexaene-31,32-diylidene]bis{bromido[(1,2,5,6-η)-cycloocta-1,5-diene]iridium(I)}, [Ir2Br2(C8H12)2(C40H46N6)], 2b. The structure of complex 2b at 100 K has orthorhombic Pbca symmetry. Each NHC moiety in 2b coordinates in a monodentate fashion to an Ir(COD) fragment. The structure exhibits disorder of the main molecule. This disorder is found in the portion of the macrocycle containing an amine group. This structure also displays C-H...Br interactions. Finally, the structure of the hexafluorophosphate salt of the imidazolin-2-ylidene-containing macrocycle, namely 5,16-dibenzyl-1λ5,5,9,12λ5,16,20-hexaazatricyclo[18.2.1.19,12]tetracosa-1(23),10,12(24),21-tetraene-1,12-diium bis(hexafluorophosphate), C32H44N62+·2PF6-, 1c, was determined. The structure of macrocycle 1c at 100 K has triclinic P-1 symmetry and was found to contain C-H...F interactions.

Pyrrole thioamide complexes of the d8 metals platinum(II), palladium(II) and gold(III)

Reactions of the cycloaurated gold(III) complexes [AuCl2(2-benzylpyridyl)] and [AuCl2(C6H4CH2NMe2)] with a set of pyrrole-2-thioamide ligands, containing various substituents on the pyrrole ring, gave a series of new gold(III) pyrrole thioamide complexes. X-ray crystal structures on two complexes indicate that the pyrrole thioamide ligand is coordinated through the deprotonated pyrrole nitrogen, as well as the sulfur atom of the deprotonated thioamide group, forming five-membered chelate ring complexes. In both complexes, the two highest trans-influence donor atoms (C and S) are mutually cis. Related sets of platinum(II) and palladium(II) pyrrole thioamide complexes were similarly prepared by reactions of cis-[PtCl2(PPh3)2] and [PdCl2(dppe)] (dppe = Ph2PCH2CH2PPh2)] respectively. The complexes were characterised by NMR and IR spectroscopies, and ESI mass spectrometry. A preliminary investigation of the activity of a selection of compounds towards A549 (adenocarcinomic human alveolar basal epithelial) cells was also carried out.

Analyses of high resolution aeromagnetic data for structural and porphyry mineral deposit mapping of the nigerian younger granite ring complexes, North - Central Nigeria

This study report the results of the different geophysical analyses carried out on the aeromagnetic data of north-central Nigeria with the object of delineating the occurrence and evaluate the structural influence on the emplacement of the porphyry mineral deposits of the Ring Complexes. The residual magnetic intensity extracted using Low Cut Guassian filter, from the high resolution aeromagnetic data of north-central Nigeria were filtered to remove high frequency near surface cultural noise and subsequently Reduced to Pole at Low Latitude (RTPLL) to focus anomaly peaks on the corresponding geological sources in order to resolve anomaly complexity associated with mid-latitude potential data. The data were further enhanced using upward continuation filter to extract desired magnetic responses at various continuation distances. Analytic Signal (AS), Centre for Exploration Techniques (CET) Grid and Porphyry Analyses were then carried out on the filtered, transformed and enhanced magnetic data to generate diagnostic magnetic attributes employed to map the boundaries of geologic magnetic sources, extract linear features and delineate the occurrence of porphyry mineral deposits across the study area.The AS responses defined twenty-four (24) arcuate/circular shaped AS anomalies which correspond to Younger Granites Ring Complexes in the northeast, northwest, southeast and central part of the study area from the low AS response Basement Complex rocks that host them. The CET grid analysis delineates several linear structures which generally trend NE – SW, NNE – SSW and ENE – WSW directions and their average length (500 m–17,500 m) and population (212–32) directly and inversely, respectively, correlate with the upward continuation distances. The CET porphyry analysis also delineates fifty (50) porphyry mineral deposits which vary in radial size from 300 m–4300 m. The occurrence of the porphyry mineral deposits within or in close proximity with the Younger Granite Ring Complexes which are either crosscut or flanked by the linear structures, suggests hydrothermal non-orogenic origin of the structurally controlled ring complexes and the associated porphyry mineralisation that result from phase mineralisation that accompanied Jurassic hydrothermal activities.

A chronic high-fat diet causes sperm head alterations in C57BL/6J mice

A chronic-positive energetic balance has been directly correlated with infertility in men, but the involved mechanisms remain unknown. Herein we investigated weather in a mouse model a chronic feeding with a diet supplemented with chicken fat affects sperm head morphology. To accomplish this, we fed mice for 16 weeks with either control food (low-fat diet, LFD) or control food supplemented with 22% chicken fat (high-fat diet, HFD). At the end of the feeding regimen, we measured: redox and inflammatory changes, cholesterol accumulation in testis and analyzed testicular morphological structure and ultra-structure and liver morphology. We found that the mice fed HFD resembled some features of the human metabolic syndrome, including systemic oxidative stress and inflammation, this group showed an increment in the following parameters; central adiposity (adiposity index: 1.07 ± 0.10 vs 2.26 ± 0.17), dyslipidemia (total cholesterol: 153.3 ± 2.6 vs 175.1 ± 8.08 mg/dL), insulin resistance (indirect Insulin resistance index, TG/HDL-c: 2.94 ± 0.33 vs 3.68 ± 0.15) and fatty liver. Increased cholesterol content measured by filipin was found in the testicles from HFD (fluorescence intensity increase to 50%), as well as an alteration of spermiogenesis. Most remarkably, a disorganized manchette-perinuclear ring complex and an altered morphology of the sperm head were observed in the spermatozoa of HFD-fed mice. These results add new information to our understanding about the mechanisms by which systemic oxidative stress and inflammation may influence sperm-head morphology and indirectly male fertility.

Lithospheric Structure Beneath the Cretaceous Chilwa Alkaline Province (CAP) in Southern Malawi and Northeastern Mozambique

AbstractWe used aeromagnetic and satellite gravity data to investigate lithospheric structure beneath the Cretaceous Chilwa Alkaline Province (CAP) in southern Malawi and adjacent Mozambique. The CAP consists of granites, syenites, nepheline syenites, basanites, phonolite dikes, and minor carbonatite bodies. The intrusions were emplaced in the Precambrian (Mesoproterozoic‐Neoproterozoic) terranes of the Southern Irumide and Mozambique orogenic belts. Aeromagnetic data show the CAP as overlapping circular anomalies typical of nested igneous ring complexes formed through caldera collapse mechanism. We used gravity data to infer that: (1) the CAP was sourced from ~30‐km wide igneous bodies now preserved in the upper crust at ~5‐km depth. (2) the CAP is underlain by up to ~45‐km thick crust (due to mafic magmatic underplating) and a lithosphere as thin as ~90 km. These data suggest that mafic magmatic underplating and lithospheric thinning occurred during a Cretaceous rifting event. We propose, based on these results and taking into account previous petrographic, geochemical, geochronological, and isotopic studies, that the silica undersaturated magmatic phase of the CAP was due to flux melting of the asthenosphere. This was followed by silica‐rich magmatic phase due to decompression melting of the asthenosphere as a result of lithospheric thinning. Lithospheric thinning and ascendance of the asthenospheric melt might have been facilitated by the presence of late Neoproterozoic to early Cambrian suture zone. The heat provided by the mafic magmatic underplating resulted in partial melting of the lower crust to form the silica‐saturated CAP intrusions from mixed magma sources.

Bi-allelic Pathogenic Variants in TUBGCP2 Cause Microcephaly and Lissencephaly Spectrum Disorders

Lissencephaly comprises a spectrum of malformations of cortical development. This spectrum includes agyria, pachygyria, and subcortical band heterotopia; each represents anatomical malformations of brain cortical development caused by neuronal migration defects. The molecular etiologies of neuronal migration anomalies are highly enriched for genes encoding microtubules and microtubule-associated proteins, and this enrichment highlights the critical role for these genes in cortical growth and gyrification. Using exome sequencing and family based rare variant analyses, we identified a homozygous variant (c.997C>T [p.Arg333Cys]) in TUBGCP2, encoding gamma-tubulin complex protein 2 (GCP2), in two individuals from a consanguineous family; both individuals presented with microcephaly and developmental delay. GCP2 forms the multiprotein γ-tubulin ring complex (γ-TuRC) together with γ-tubulin and other GCPs to regulate the assembly of microtubules. By querying clinical exome sequencing cases and through GeneMatcher-facilitated collaborations, we found three additional families with bi-allelic variation and similarly affected phenotypes including a homozygous variant (c.1843G>C [p.Ala615Pro]) in two families and compound heterozygous variants consisting of one missense variant (c.889C>T [p.Arg297Cys]) and one splice variant (c.2025-2A>G) in another family. Brain imaging from all five affected individuals revealed varying degrees of cortical malformations including pachygyria and subcortical band heterotopia, presumably caused by disruption of neuronal migration. Our data demonstrate that pathogenic variants in TUBGCP2 cause an autosomal recessive neurodevelopmental trait consisting of a neuronal migration disorder, and our data implicate GCP2 as a core component of γ-TuRC in neuronal migrating cells.

Synthesis and structural characterization of nickel(II) complexes of 20-membered macrocyclic rings bearing chelating bis(N-heterocyclic carbene) ligands

20-Membered macrocyclic rings (Lbenz and Limid) containing two amine moieties and two N-heterocyclic carbene (NHC) moieties coordinate in a κ2 coordination mode to Ni(II) through the NHC moieties. Complexes 3a and 3b were synthesized by generating free carbenes in situ from bis-benzimidazolium bromide salts and subsequent addition of one equivalent of [Ni(DME)X2] (X = Cl, Br) at 25 °C. Free carbenes of 20-membered macrocyclic ligands Lbenz and Limid can be generated in situ in THF-d8 and characterized by 1H and 13C NMR spectroscopy. 3b can also be synthesized by treatment of silver(I)-bis(NHC) complex 2a with one equivalent of [NiBr2(DME)] in refluxing methylene chloride for 20 h affording yields of up to70%. Complexes 3a and 3b were characterized by X-ray diffraction. [NiI2(Lbenz)] 5c was prepared by halogen exchange of 3a or 3b with NaI at 25 °C. Ni complexes of Limid can be prepared and characterized in situ but cannot be isolated cleanly from the reaction mixtures. The cis and trans isomers of [NiCl2(Limid)] (4a and 4b) and the bromide derivative 4c were characterized by X-ray crystallography by judicious selection of individual, high-quality crystals obtained from crude reaction mixtures.

α-Fodrin is required for the organization of functional microtubules during mitosis

ABSTRACTThe cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin’s involvement in cancer.

Co-expression of CCT subunits hints at TRiC assembly.

The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin)with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.

Direct observation of branching MT nucleation in living animal cells.

Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.