How Microtubules Build the Spindle Branch by Branch.

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

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 ∼3.8 Å resolution, revealing an asymmetric, 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 (>100,000 Å2) 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.

Mechanisms of Mitotic Spindle Assembly.

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

Microtubule-Organizing Centers.

The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems.

Microtubule nucleation for spindle assembly: one molecule at a time.

The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.

Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

The structure of the γ-TuRC at the microtubule minus end - not just one solution.

In cells, microtubules (MTs) assemble from α/ß-tubulin subunits at nucleation sites containing the γ-tubulin ring complex (γ-TuRC). Within the γ-TuRC, exposed γ-tubulin molecules act as templates for MT assembly by interacting with α/ß-tubulin. The vertebrate γ-TuRC is scaffolded by γ-tubulin-interacting proteins GCP2-6 arranged in a specific order. Interestingly, the γ-tubulin molecules in the γ-TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ-TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ-TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α-tubulin mutant, resulted in a closed, cylindrical γ-TuRC showing canonical interactions between all γ-tubulin molecules and α/ß-tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ-TuRC, with some γ-tubulin molecules failing to align with α/ß-tubulin. This review discusses these outcomes, along with the broader implications.

A germline-specific role for unconventional components of the γ-tubulin complex in Caenorhabditis elegans.

The γ-tubulin complex (γTuC) is a widely conserved microtubule nucleator, but some of its components, namely GCP4, GCP5 and GCP6 (also known as TUBGCP4, TUBGCP5 and TUBGCP6, respectively), have not been detected in Caenorhabditis elegans. Here, we identified two γTuC-associated proteins in C. elegans, GTAP-1 and GTAP-2, for which apparent orthologs were detected only in the genus Caenorhabditis. GTAP-1 and GTAP-2 were found to localize at centrosomes and the plasma membrane of the germline, and their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved γTuC component MZT-1 (also known as MOZART1 and MZT1) was essential for the localization of centrosomal γ-tubulin, depletion of GTAP-1 and/or GTAP-2 caused up to 50% reduction of centrosomal γ-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTAP-2 contributed to efficient recruitment of the γTuC to the plasma membrane. Depletion of GTAP-1, but not of GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure of the adult germline. We propose that GTAP-1 and GTAP-2 are unconventional components of the γTuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the γTuC to specific subcellular sites in a tissue-specific manner.

Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function.

Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes [known as spindle pole bodies (SPBs)] are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.

Actin filaments form a size-dependent diffusion barrier around centrosomes.

The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actin-based diffusion barrier gates microtubule nucleation by interfering with γ-tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size-dependent manner.

c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation.

Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics.

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of γ-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.

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.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

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.

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.

Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein Samp1.

We have investigated a possible role for the inner nuclear membrane protein Samp1 (also known as TMEM201) in the mitotic machinery. Live-cell imaging showed that Samp1a-YFP (Samp1a is the short isoform of Samp1) distributed as filamentous structures in the mitotic spindle, partially colocalising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistent with this, mitotic spindles in Samp1-depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes that were rescued by overexpression of Samp1a-YFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and the HAUS6 subunit of the Augmin complex in live cells. The levels of HAUS6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of HAUS6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

γ-Tubulin interacts with E2F transcription factors to regulate proliferation and endocycling in Arabidopsis.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

Centrosome Abnormalities and Polyploidy in Murine Mammary Carcinomas with Different Degrees of Hormone Responsiveness.

Centrosome amplification leads to aberrant mitosis, giving rise to aneuploidy and it has been associated with poor prognosis in human cancers. This study aimed to evaluate the relationship between polyploidy, centrosome abnormalities, and response to endocrine treatment in progestin-induced mouse mammary carcinomas. We found cells with three or more centrosomes in the polyploid tumors. The endocrine unresponsive tumors showed a higher average number of centrosomes per cell than the responsive tumors. The results suggest an association between polyploidy and centrosome amplification with the resistance to endocrine therapy in this luminal breast cancer model.

Cytoplasmic MTOCs control spindle orientation for asymmetric cell division in plants.

Proper orientation of the cell division axis is critical for asymmetric cell divisions that underpin cell differentiation. In animals, centrosomes are the dominant microtubule organizing centers (MTOC) and play a pivotal role in axis determination by orienting the mitotic spindle. In land plants that lack centrosomes, a critical role of a microtubular ring structure, the preprophase band (PPB), has been observed in this process; the PPB is required for orienting (before prophase) and guiding (in telophase) the mitotic apparatus. However, plants must possess additional mechanisms to control the division axis, as certain cell types or mutants do not form PPBs. Here, using live imaging of the gametophore of the moss Physcomitrella patens, we identified acentrosomal MTOCs, which we termed "gametosomes," appearing de novo and transiently in the prophase cytoplasm independent of PPB formation. We show that gametosomes are dispensable for spindle formation but required for metaphase spindle orientation. In some cells, gametosomes appeared reminiscent of the bipolar MT "polar cap" structure that forms transiently around the prophase nucleus in angiosperms. Specific disruption of the polar caps in tobacco cells misoriented the metaphase spindles and frequently altered the final division plane, indicating that they are functionally analogous to the gametosomes. These results suggest a broad use of transient MTOC structures as the spindle orientation machinery in plants, compensating for the evolutionary loss of centrosomes, to secure the initial orientation of the spindle in a spatial window that allows subsequent fine-tuning of the division plane axis by the guidance machinery.