CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC.

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.

The AP-2 complex interacts with γ-TuRC and regulates the proliferative capacity of neural progenitors.

Centrosomes are organelles that nucleate microtubules via the activity of gamma-tubulin ring complexes (γ-TuRC). In the developing brain, centrosome integrity is central to the progression of the neural progenitor cell cycle, and its loss leads to microcephaly. We show that NPCs maintain centrosome integrity via the endocytic adaptor protein complex-2 (AP-2). NPCs lacking AP-2 exhibit defects in centrosome formation and mitotic progression, accompanied by DNA damage and accumulation of p53. This function of AP-2 in regulating the proliferative capacity of NPCs is independent of its role in clathrin-mediated endocytosis and is coupled to its association with the GCP2, GCP3, and GCP4 components of γ-TuRC. We find that AP-2 maintains γ-TuRC organization and regulates centrosome function at the level of MT nucleation. Taken together, our data reveal a novel, noncanonical function of AP-2 in regulating the proliferative capacity of NPCs and open new avenues for the identification of novel therapeutic strategies for the treatment of neurodevelopmental and neurodegenerative disorders with AP-2 complex dysfunction.

Proteomic profiling of centrosomes across multiple mammalian cell and tissue types by an affinity capture method.

Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis.

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.

The minus-end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules.

During mitosis, microtubules in the spindle turn over continuously. At spindle poles, where microtubule minus ends are concentrated, microtubule nucleation and depolymerization, the latter required for poleward microtubule flux, happen side by side. How these seemingly antagonistic processes of nucleation and depolymerization are coordinated is not understood. Here, we reconstitute this coordination in vitro combining different pole-localized activities. We find that the spindle pole-localized kinesin-13 KIF2A is a microtubule minus-end depolymerase, in contrast to its paralog MCAK. Due to its asymmetric activity, KIF2A still allows microtubule nucleation from the γ-tubulin ring complex (γTuRC), which serves as a protective cap shielding the minus end against KIF2A binding. Efficient γTuRC uncapping requires the combined action of KIF2A and a microtubule severing enzyme, leading to treadmilling of the uncapped microtubule driven by KIF2A. Together, these results provide insight into the molecular mechanisms by which a minimal protein module coordinates microtubule nucleation and depolymerization at spindle poles consistent with their role in poleward microtubule flux.

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.

Live-cell imaging for analysis of the NK cell immunological synapse.

The immunological synapse (IS) between NK cells and cancer cells is instrumental for the initiation of tumor-specific cytotoxicity. Improper function of processes at the IS can lead to NK cell unresponsiveness, contributing to tumor immune escape. Critical steps at the IS include target cell recognition, conjugation of NK cell and cancer cell, cytotoxic granule convergence to the microtubule-organizing center (MTOC), granule polarization to the IS, and degranulation. Here, we describe confocal live-cell imaging methods for the analysis of these processes at the immunological synapse, with a focus on mechanisms of cancer cell resistance facilitating escape from NK cell cytotoxicity.

Physcomitrium patens SUN2 Mediates MTOC Association with the Nuclear Envelope and Facilitates Chromosome Alignment during Spindle Assembly.

Plant cells lack centrosomes and instead utilize acentrosomal microtubule organizing centers (MTOCs) to rapidly increase the number of microtubules at the onset of spindle assembly. Although several proteins required for MTOC formation have been identified, how the MTOC is positioned at the right place is not known. Here, we show that the inner nuclear membrane protein SUN2 is required for MTOC association with the nuclear envelope (NE) during mitotic prophase in the moss Physcomitrium patens. In actively dividing protonemal cells, microtubules accumulate around the NE during prophase. In particular, regional MTOC is formed at the apical surface of the nucleus. However, microtubule accumulation around the NE was impaired and apical MTOCs were mislocalized in sun2 knockout cells. Upon NE breakdown, the mitotic spindle was assembled with mislocalized MTOCs. However, completion of chromosome alignment in the spindle was delayed; in severe cases, the chromosome was transiently detached from the spindle body. SUN2 tended to localize to the apical surface of the nucleus during prophase in a microtubule-dependent manner. Based on these results, we propose that SUN2 facilitates the attachment of microtubules to chromosomes during spindle assembly by localizing microtubules to the NE. MTOC mispositioning was also observed during the first division of the gametophore tissue. Thus, this study suggests that microtubule-nucleus linking, a well-known function of SUN in animals and yeast, is conserved in plants.

Mapping of centriolar proteins onto the post-embryonic lineage of C. elegans.

Centrioles, together with the surrounding peri-centriolar material (PCM), constitute the centrosome, a major microtubule-organizing center of animal cells. Despite being critical in many cells for signaling, motility and division, centrioles can be eliminated in some systems, including in the vast majority of differentiating cells during embryogenesis in Caenorhabditis elegans. Whether the cells retaining centrioles in the resulting L1 larvae do so because they lack an activity that eliminates centrioles in the other cells is not known. Moreover, the extent to which centrioles and PCM remain present in later stages of worm development, when all cells but those of the germ line are terminally differentiated, is not known. Here, by fusing cells that lack centrioles with cells that retain them, we established that L1 larvae do not possess a diffusible elimination activity sufficient to remove centrioles. Moreover, analyzing PCM core proteins in L1 larval cells that retain centrioles, we found that some such proteins, but not all, are present as well. Furthermore, we uncovered that foci of centriolar proteins remain present in specific terminally differentiated cells of adult hermaphrodites and males, in particular in the somatic gonad. Correlating the time at which cells were born with the fate of their centrioles revealed that it is not cell age, but instead cell fate, that determines whether and when centrioles are eliminated. Overall, our work maps the localization of centriolar and PCM core proteins in the post-embryonic C. elegans lineage, thereby providing an essential blueprint for uncovering mechanisms modulating their presence and function.

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.

Autoinhibitory mechanism controls binding of centrosomin motif 1 to γ-tubulin ring complex.

The γ-tubulin ring complex (γTuRC) is the principal nucleator of cellular microtubules, and the microtubule-nucleating activity of the complex is stimulated by binding to the γTuRC-mediated nucleation activator (γTuNA) motif. The γTuNA is part of the centrosomin motif 1 (CM1), which is widely found in γTuRC stimulators, including CDK5RAP2. Here, we show that a conserved segment within CM1 binds to the γTuNA and blocks its association with γTuRCs; therefore, we refer to this segment as the γTuNA inhibitor (γTuNA-In). Mutational disruption of the interaction between the γTuNA and the γTuNA-In results in a loss of autoinhibition, which consequently augments microtubule nucleation on centrosomes and the Golgi complex, the two major microtubule-organizing centers. This also causes centrosome repositioning, leads to defects in Golgi assembly and organization, and affects cell polarization. Remarkably, phosphorylation of the γTuNA-In, probably by Nek2, counteracts the autoinhibition by disrupting the γTuNA‒γTuNA-In interaction. Together, our data reveal an on-site mechanism for controlling γTuNA function.

Control of centrosome distal appendages assembly and disassembly.

Centrosomes are microtubule organizing centers involved in chromosome segregation, spindle orientation, cell motility and cilia formation. In recent years, they have also emerged as key modulators of asymmetric cell division. Centrosomes are composed of two centrioles that initiate duplication in S phase. The conservative nature of centriole duplication means that the two centrioles of a G1 cell are of different ages. They are also structurally different as only the older centriole carries appendages, an assembly of a subset of proteins primarily required for cilia formation. In a growing tissue, the non-motile, primary cilium acts as a mechano- and sensory organelle that influences cell behavior via modulation of signaling pathways. Here, we discuss the most recent findings about distal appendage composition and function, as well as cell cycle-specific regulation and their implications in various diseases.

The many faces of the bouquet centrosome MTOC in meiosis and germ cell development.

Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.

Microtubule cytoskeleton: The centrosome gains a membrane.

Identification of a membrane structure, termed the 'centriculum', in Caenorhabditis elegans embryos challenges the textbook view of the centrosome - a major microtubule organizing center in animal cells - as an organelle that lacks a surrounding membrane.

A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.

The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.

Microtubule nucleation and γTuRC centrosome localization in interphase cells require ch-TOG.

Organization of microtubule arrays requires spatio-temporal regulation of the microtubule nucleator γ-tubulin ring complex (γTuRC) at microtubule organizing centers (MTOCs). MTOC-localized adapter proteins are thought to recruit and activate γTuRC, but the molecular underpinnings remain obscure. Here we show that at interphase centrosomes, rather than adapters, the microtubule polymerase ch-TOG (also named chTOG or CKAP5) ultimately controls γTuRC recruitment and activation. ch-TOG co-assembles with γTuRC to stimulate nucleation around centrioles. In the absence of ch-TOG, γTuRC fails to localize to these sites, but not the centriole lumen. However, whereas some ch-TOG is stably bound at subdistal appendages, it only transiently associates with PCM. ch-TOG's dynamic behavior requires its tubulin-binding TOG domains and a C-terminal region involved in localization. In addition, ch-TOG also promotes nucleation from the Golgi. Thus, at interphase centrosomes stimulation of nucleation and γTuRC attachment are mechanistically coupled through transient recruitment of ch-TOG, and ch-TOG's nucleation-promoting activity is not restricted to centrosomes.

Septum-associated microtubule organizing centers within conidia support infectious development by the blast fungus Magnaporthe oryzae.

Cytoplasmic microtubule arrays play important and diverse roles within fungal cells, including serving as molecular highways for motor-driven organelle motility. While the dynamic plus ends of cytoplasmic microtubules are free to explore the cytoplasm through their stochastic growth and shrinkage, their minus ends are nucleated at discrete organizing centers, composed of large multi-subunit protein complexes. The location and composition of these microtubule organizing centers varies depending on genus, cell type, and in some instances cell-cycle stage. Despite their obvious importance, our understanding of the nature, diversity, and regulation of microtubule organizing centers in fungi remains incomplete. Here, using three-color fluorescence microscopy based live-cell imaging, we investigate the organization and dynamic behavior of the microtubule cytoskeleton within infection-related cell types of the filamentous fungus,Magnaporthe oryzae, a highly destructive pathogen of rice and wheat. We provide data to support the idea that cytoplasmic microtubules are nucleated at septa, rather than at nuclear spindle pole bodies, within the three-celled blast conidium, and provide new insight into remodeling of the microtubule cytoskeleton during nuclear division and inheritance. Lastly, we provide a more complete picture of the architecture and subcellular organization of the prototypical blast appressorium, a specialized pressure-generating cell type used to invade host tissue. Taken together, our study provides new insight into microtubule nucleation, organization, and dynamics in specialized and differentiated fungal cell types.

The centriolar tubulin code.

Centrioles are microtubule-based cell organelles present in most eukaryotes. They participate in the control of cell division as part of the centrosome, the major microtubule-organizing center of the cell, and are also essential for the formation of primary and motile cilia. During centriole assembly as well as across its lifetime, centriolar tubulin display marks defined by post-translational modifications (PTMs), such as glutamylation or acetylation. To date, the functions of these PTMs at centrioles are not well understood, although pioneering experiments suggest a role in the stability of this organelle. Here, we review the current knowledge regarding PTMs at centrioles with a particular focus on a possible link between these modifications and centriole's architecture, and propose possible hypothesis regarding centriolar tubulin PTMs's function.