Loss of ninein interferes with osteoclast formation and causes premature ossification.

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human NINEIN gene have been linked to Seckel syndrome and to a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice, and that its absence reduces the fusion of precursor cells into syncytial osteoclasts, whereas the number of osteoblasts remains unaffected. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold.

Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease.

Microtubule nucleation without a ring?

The native γ-tubulin ring complex is an asymmetric, imperfect template for microtubule nucleation. Wieczorek et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202009146) and Zimmermann et al. (2020. Sci. Adv.https://doi.org/10.1126/sciadv.abe0894) have reconstituted a recombinant complex that allows study of structure-function relationships and regulatory mechanisms.

A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes.

γ-Tubulin is the main protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (γ-tubulin complex proteins): γ-tubulin small complexes (γTuSCs) that contain γ-tubulin, and GCPs 2 and 3; and γ-tubulin ring complexes (γTuRCs) that contain multiple γTuSCs in addition to GCPs 4, 5 and 6. Whereas the structure and assembly properties of γTuSCs have been intensively studied, little is known about the assembly of γTuRCs and the specific roles of GCPs 4, 5 and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex within the γTuRC that assembles independently of the presence of γTuSCs. Incubation of this sub-complex with cytoplasmic extracts containing γTuSCs leads to the reconstitution of γTuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. We also demonstrate that these are involved in the assembly or stabilization of the γTuRC.

Epidermal development requires ninein for spindle orientation and cortical microtubule organization.

In mammalian skin, ninein localizes to the centrosomes of progenitor cells and relocates to the cell cortex upon differentiation of keratinocytes, where cortical arrays of microtubules are formed. To examine the function of ninein in skin development, we use epidermis-specific and constitutive ninein-knockout mice to demonstrate that ninein is necessary for maintaining regular protein levels of the differentiation markers filaggrin and involucrin, for the formation of desmosomes, for the secretion of lamellar bodies, and for the formation of the epidermal barrier. Ninein-deficient mice are viable but develop a thinner skin with partly impaired epidermal barrier. We propose two underlying mechanisms: first, ninein contributes to spindle orientation during the division of progenitor cells, whereas its absence leads to misoriented cell divisions, altering the pool of progenitor cells. Second, ninein is required for the cortical organization of microtubules in differentiating keratinocytes, and for the cortical re-localization of microtubule-organizing proteins, and may thus affect any mechanisms that depend on localized microtubule-dependent transport.

Stabilization of microtubules restores barrier function after cytokine-induced defects in reconstructed human epidermis.

BACKGROUND: A variety of human skin disorders is characterized by defects in the epidermal barrier, leading to dehydration, itchiness, and rashes. Previously published literature suggests that microtubule stabilization at the cortex of differentiating keratinocytes is necessary for the formation of the epidermal barrier. OBJECTIVES: We tested whether stabilization of microtubules with paclitaxel or epothilone B can repair barrier defects that were experimentally induced in three-dimensional culture models of epidermis. METHODS: We established two models of defective epidermis in vitro, using three-dimensional cultures of primary human keratinocytes on filter supports: immature reconstructed human epidermis (RHE), and RHE that was compromised by treatment with inflammatory cytokines, the latter mimicking defects seen in atopic dermatitis. RESULTS: Both paclitaxel and epothilone B promoted keratinocyte differentiation, accumulation of junctional proteins at the cell cortex, and the early appearance of lamellar bodies in immature RHE, whereas destabilization of microtubules by nocodazole had the reverse effect. Moreover, stabilization of microtubules rescued the barrier after cytokine treatment. The rescued barrier function correlated with the restoration of filaggrin and loricrin protein levels, the cortical accumulation of junctional proteins (E-cadherin, ß-catenin, and claudin-1), and with the secretion of lamellar bodies. CONCLUSIONS: Our data suggest that the microtubule network is important for the formation of the epidermis, and that stabilization of microtubules promotes barrier formation. Microtubule stabilization may support regeneration of damaged skin, by restoring or improving the barrier.

Assembly and regulation of γ-tubulin complexes.

Microtubules are major constituents of the cytoskeleton in all eukaryotic cells. They are essential for chromosome segregation during cell division, for directional intracellular transport and for building specialized cellular structures such as cilia or flagella. Their assembly has to be controlled spatially and temporally. For this, the cell uses multiprotein complexes containing γ-tubulin. γ-Tubulin has been found in two different types of complexes, γ-tubulin small complexes and γ-tubulin ring complexes. Binding to adaptors and activator proteins transforms these complexes into structural templates that drive the nucleation of new microtubules in a highly controlled manner. This review discusses recent advances on the mechanisms of assembly, recruitment and activation of γ-tubulin complexes at microtubule-organizing centres.

Centriolar satellites prevent uncontrolled degradation of centrosome proteins: a speculative review.

Centriolar satellites are small electron-dense structures in the cytoplasm, mostly surrounding the pericentriolar material. Initially viewed as shuttles for the transport of centrosomal proteins, they have been implicated in the assembly of the pericentriolar material and in ciliogenesis. Although numerous proteins have been identified as components of centriolar satellites, their molecular function remains unclear. In this review article, we discuss recent findings that characterize centriolar satellites as regulators of protein degradation pathways: by sequestering E3 ligase MIB1, deacetylase HDAC6, and proteins of the autophagy pathway, centriolar satellites may regulate the turnover of centrosomal and ciliary components, protecting them from removal via proteasomal degradation, autophagy, and aggresomes.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

Nuclear alignment in myotubes requires centrosome proteins recruited by nesprin-1.

Myotubes are syncytial cells generated by fusion of myoblasts. Among the numerous nuclei in myotubes of skeletal muscle fibres, the majority are equidistantly positioned at the periphery, except for clusters of multiple nuclei underneath the motor endplate. The correct positioning of nuclei is thought to be important for muscle function and requires nesprin-1 (also known as SYNE1), a protein of the nuclear envelope. Consistent with this, mice lacking functional nesprin-1 show defective nuclear positioning and present aspects of Emery-Dreifuss muscular dystrophy. In this study, we perform small interfering RNA (siRNA) experiments in C2C12 myoblasts undergoing differentiation, demonstrating that the positioning of nuclei requires PCM-1, a protein of the centrosome that relocalizes to the nuclear envelope at the onset of differentiation in a manner that is dependent on the presence of nesprin-1. PCM-1 itself is required for recruiting proteins of the dynein-dynactin complex and of kinesin motor complexes. This suggests that microtubule motors that are attached to the nuclear envelope support the movement of nuclei along microtubules, to ensure their correct positioning in the myotube.

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains.

Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Mutations in TUBGCP4 alter microtubule organization via the γ-tubulin ring complex in autosomal-recessive microcephaly with chorioretinopathy.

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.

γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning.

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, we propose a role for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs.

A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis.

Centriolar satellites are small, granular structures that cluster around centrosomes, but whose biological function and regulation are poorly understood. We show that centriolar satellites undergo striking reorganization in response to cellular stresses such as UV radiation, heat shock, and transcription blocks, invoking acute and selective displacement of the factors AZI1/CEP131, PCM1, and CEP290 from this compartment triggered by activation of the stress-responsive kinase p38/MAPK14. We demonstrate that the E3 ubiquitin ligase MIB1 is a new component of centriolar satellites, which interacts with and ubiquitylates AZI1 and PCM1 and suppresses primary cilium formation. In response to cell stress, MIB1 is abruptly inactivated in a p38-independent manner, leading to loss of AZI1, PCM1, and CEP290 ubiquitylation and concomitant stimulation of ciliogenesis, even in proliferating cells. Collectively, our findings uncover a new two-pronged signalling response, which by coupling p38-dependent phosphorylation with MIB1-catalysed ubiquitylation of ciliogenesis-promoting factors plays an important role in controlling centriolar satellite status and key centrosomal functions in a cell stress-regulated manner.

Virtual and biophysical screening targeting the γ-tubulin complex--a new target for the inhibition of microtubule nucleation.

Microtubules are the main constituents of mitotic spindles. They are nucleated in large amounts during spindle assembly, from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). With the aim of developing anti-cancer drugs targeting these nucleating complexes, we analyzed the interface between GCP4 and γ-tubulin proteins usually located in a multiprotein complex named γ-TuRC (γ-Tubulin Ring Complex). 10 ns molecular dynamics simulations were performed on the heterodimers to obtain a stable complex in silico and to analyze the residues involved in persistent protein-protein contacts, responsible for the stability of the complex. We demonstrated in silico the existence of a binding pocket at the interface between the two proteins upon complex formation. By combining virtual screening using a fragment-based approach and biophysical screening, we found several small molecules that bind specifically to this pocket. Sub-millimolar fragments have been experimentally characterized on recombinant proteins using differential scanning fluorimetry (DSF) for validation of these compounds as inhibitors. These results open a new avenue for drug development against microtubule-nucleating γ-tubulin complexes.

Assembly of gamma-tubulin ring complexes: implications for cell biology and disease.

Microtubules are among the main constituents of the cytoskeleton. They are assembled from dimers of alpha- and beta-tubulin. This assembly occurs preferentially at organizing centers such as the centrosomes, catalyzed by multiprotein complexes of gamma-tubulin. At the beginning of mitosis, the amount of gamma-tubulin complexes at the centrosomes increases sharply, supporting the sudden formation of numerous spindle microtubules. Recent studies on the structure of gamma-tubulin complex proteins have advanced our understanding of the assembly process of gamma-tubulin complexes, and have pointed toward putative mechanisms of microtubule nucleation. Moreover, the discovery of novel proteins associated with gamma-tubulin complexes has illustrated the possibilities of how gamma-tubulin might be recruited and regulated at specific sites of microtubule organization. This chapter highlights recent developments in the field and discusses the potential of the gamma-tubulin complex as a pharmacological target, to control proliferation of cells.

Microtubule nucleation by γ-tubulin complexes.

Microtubule nucleation is regulated by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ-tubulin γTuRC component (γ-tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation.

Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation.

Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity.

A CaMKIIß signaling pathway at the centrosome regulates dendrite patterning in the brain.

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and ß isoforms in the brain. Although CaMKIIα functions have been elucidated, the isoform-specific catalytic functions of CaMKIIß have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIß operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKIIß to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20-anaphase promoting complex) as a centrosomal substrate of CaMKIIß. CaMKIIß phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKIIß that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.