Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer.

DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein.

Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent Km values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 µM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC50 value of 5.1 ± 0.5 µM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC50 value of 2.6 ± 0.6 µM.

Facile purification of active recombinant mouse cytosolic carboxypeptidase 6 from Escherichia coli.

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.

RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control.

The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.

Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.

AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('ß1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.

Differential activation mechanisms of lipid GPCRs by lysophosphatidic acid and sphingosine 1-phosphate.

Lysophospholipids are bioactive lipids and can signal through G-protein-coupled receptors (GPCRs). The best studied lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). The mechanisms of lysophospholipid recognition by an active GPCR, and the activations of lysophospholipid GPCR-G-protein complexes remain unclear. Here we report single-particle cryo-EM structures of human S1P receptor 1 (S1P1) and heterotrimeric Gi complexes formed with bound S1P or the multiple sclerosis (MS) treatment drug Siponimod, as well as human LPA receptor 1 (LPA1) and Gi complexes in the presence of LPA. Our structural and functional data provide insights into how LPA and S1P adopt different conformations to interact with their cognate GPCRs, the selectivity of the homologous lipid GPCRs for S1P versus LPA, and the different activation mechanisms of these GPCRs by LPA and S1P. Our studies also reveal specific optimization strategies to improve the MS-treating S1P1-targeting drugs.

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry.

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

Non-covalent SARS-CoV-2 Mpro inhibitors developed from in silico screen hits.

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.

Development and characterization of functional antibodies targeting NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

Olfactomedin-4 (OLFM4) is an olfactomedin-domain-containing glycoprotein, which regulates cell adhesion, proliferation, gastrointestinal inflammation, innate immunity and cancer metastasis. In the present study we investigated its role in skin regeneration. We found that OLFM4 expression is transiently upregulated in the proliferative phase of cutaneous wound healing in humans as well as in mice. Moreover, a significant increase in OLFM4 expression was detected in the skin of lesional psoriasis, a chronic inflammatory disease characterized by keratinocyte hyperproliferation. In vitro experiments demonstrated that OLFM4 selectively stimulated keratinocyte proliferation and increased both keratinocyte and fibroblast migration. Using proteotranscriptomic pathway analysis we revealed that transcription factors POU5F1/OCT4 and ESR1 acted as hubs for OLFM4-induced signalling in keratinocytes. In vivo experiments utilizing mouse splinted full-thickness cutaneous wound healing model showed that application of recombinant OLFM4 protein can significantly improve wound healing efficacy. Taken together, our results suggest that OLFM4 acts as a transiently upregulated inflammatory signal that promotes wound healing by regulating both dermal and epidermal cell compartments of the skin.

(More than) doubling down: Effective fibrinolysis at a reduced rt-PA dose for catheter-directed thrombolysis combined with histotripsy.

Deep vein thrombosis is a major source of morbidity and mortality worldwide. For acute proximal deep vein thrombosis, catheter-directed thrombolytic therapy is an accepted method for vessel recanalization. Thrombolytic therapy is not without risk, including the potential for hemorrhagic bleeding that increases with lytic dose. Histotripsy is a focused ultrasound therapy that generates bubble clouds spontaneously in tissue at depth. The mechanical activity of histotripsy increases the efficacy of thrombolytic therapy at doses consistent with current pharmacomechanical treatments for venous thrombosis. The objective of this study was to determine the influence of lytic dose on histotripsy-enhanced fibrinolysis. Human whole blood clots formed in vitro were exposed to histotripsy and a thrombolytic agent (recombinant tissue plasminogen activator, rt-PA) in a venous flow model perfused with plasma. Lytic was administered into the clot via an infusion catheter at concentrations ranging from 0 (control) to 4.54 µg/mL (a common clinical dose for catheter-directed thrombolysis). Following treatment, perfusate samples were assayed for markers of fibrinolysis, hemolysis, and intact red blood cells and platelets. Fibrinolysis was equivalent between the common clinical dose of rt-PA (4.54 µg/mL) and rt-PA at a reduction to one-twentieth of the common clinical dose (0.23 µg/mL) when combined with histotripsy. Minimal changes were observed in hemolysis for treatment arms with or without histotripsy, potentially due to clot damage from insertion of the infusion catheter. Likewise, histotripsy did not increase the concentration of red blood cells or platelets in the perfusate following treatment compared to rt-PA alone. At the highest lytic dose, a refined histotripsy exposure scheme was implemented to cover larger areas of the clot. The updated exposure scheme improved clot mass loss and fibrinolysis relative to administration of lytic alone. Overall, the data collected in this study indicate the rt-PA dose can be reduced by more than a factor of ten and still promote fibrinolysis when combined with histotripsy.

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications.

Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.

N-Substituted 5-(1H-Indol-2-yl)-2-methoxyanilines Are Allosteric Inhibitors of the Linoleate Oxygenase Activity of Selected Mammalian ALOX15 Orthologs: Mechanism of Action.

Here, we describe the first systematic study on the mechanism of substrate-selective inhibition of mammalian ALOX15 orthologs. For this purpose, we prepared a series of N-substituted 5-(1H-indol-2-yl)anilines and found that (N-(5-(1H-indol-2-yl)-2-methoxyphenyl)sulfamoyl)carbamates and their monofluorinated analogues are potent and selective inhibitors of the linoleate oxygenase activity of rabbit and human ALOX15. Introduction of a 2-methoxyaniline moiety into the core pharmacophore plays a crucial role in substrate-selective inhibition of ALOX15-catalyzed oxygenation of linoleic acid at submicromolar concentrations without affecting arachidonic acid oxygenation. Steady-state kinetics, mutagenesis studies, and molecular dynamics (MD) simulations suggested an allosteric mechanism of action. Using a dimer model of ALOX15, our MD simulations suggest that the binding of the inhibitor at the active site of one monomer induces conformational alterations in the other monomer so that the formation of a productive enzyme-linoleic acid complex is energetically compromised.

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.