Microtubule-organizing Center Research Articles

Single-cell proteo-genomic reveals a comprehensive map of centrosome-associated spliceosome components

Ribonucleoprotein (RNP) condensates are crucial for controlling RNA metabolism and splicing events in animal cells. We used spatial proteomics and transcriptomic to elucidate RNP interaction networks at the centrosome, the main microtubule-organizing center in animal cells. We found a number of cell-type specific centrosome-associated spliceosome interactions localized in subcellular structures involved in nuclear division and ciliogenesis. A component of the nuclear spliceosome BUD31 was validated as an interactor of the centriolar satellite protein OFD1. Analysis of normal and disease cohorts identified the cholangiocarcinoma as target of centrosome-associated spliceosome alterations. Multiplexed single-cell fluorescent microscopy for the centriole linker CEP250 and spliceosome components including BCAS2, BUD31, SRSF2 and DHX35 recapitulated bioinformatic predictions on the centrosome-associated spliceosome components tissue-type specific composition. Collectively, centrosomes and cilia act as anchor for cell-type specific spliceosome components, and provide a helpful reference for explore cytoplasmic condensates functions in defining cell identity and in the origin of rare diseases.

Abstract 2116: Phosphorylated extracellular signal-regulated kinase (pERK) activation in T effector cells as a target engagement biomarker for the DGKζ inhibitor BAY2965501 in clinical trials

Abstract Introduction: BAY2965501 is a small molecule that is a potent selective inhibitor of diacylglycerol kinases zeta (DGKζ). DGK are a family of enzymes that catalyzes the phosphorylation of the membrane lipid sn-1,2 diacylglycerol (DAG) to form phosphatidic acid. In T-cells, DGKζ is considered to play a key role in T-cell receptor (TCR) signaling. One of the signaling pathways controls T-cell proliferation and activation in response to antigen receptor engagement is controlled by extracellular-regulated kinase ERK1 and ERK2 (ERK1/2). The RASGRP/ERK pathway plays a crucial part the polarization of microtubule-organizing center in the cytotoxic T-cells, which is required to deliver granzyme to the targeted cells. Restoring T-cell cellular DAG level and activation of ERK1/2 in anergic T-cells via DGK inhibition has been considered a viable mechanism to assist T-cell mediate tumor cell kill. BAY2965501 showed that pERK could be activated in preclinical models. Phosphorylation of ERK (pERK) inhibition in tumor cells and its difficulty in clinical implementation has been largely acknowledged in oncology community. Circulating T-cell pERK changes as a target engagement biomarker may provide a plausible solution in clinical trial. Here, we report a development of a fit-for-purpose pERK+CD8+ assay. Method: Feasibilities to detect BAY2965501 induced pERK activation was tested to minimize the expected clinical hurdles by using various conditions such as various blood collection tubes (BCT), CD3/CD28 concentrations, and incubation times were evaluated. Whole blood was stimulated with BAY2965501 followed by activation with anti-CD3/anti-CD28 cocktail with subsequent lysis and fixation procedure including freezing. Samples were then stained with antibodies against T-cell cell-surface markers and anti-pERK antibody followed by analyzing on a flow cytometer. Results: A pERK+CD8+ cell frequency increase was successfully observed after stimulation with BAY2965501 at different concentrations (e.g., 12.89% spiked vs. 30.42% non-spiked). A peak of pERK+CD8+ cell frequency was observed with incubation of anti-CD3/CD28 at 15 min compared to 5 min or 30 min. Titration of anti-CD3/anti-CD28 cocktail resulted in saturation observed at a concentration of 2 µg/mL (range 1-4 µg/ml). Three collection tubes were tested; 1) Sodium heparin (SoHep); 2) Citrate and 3) TruCulture. SoHep showed the highest pERK+ CD8+ cell frequency (8.29%) compared to Citrate BCT or TruCulture. Activated and stimulated samples are stable at -80°C for at least 6 days (D0 30.42% vs D6-80°C 29.52%) although samples are not stable at ambient or cooled conditions (D0 12.38% vs D1RT 3.02% vs D14°C 7.66%). Conclusion: These findings support implementing the pERK+CD8+ assay in T cells as a target engagement biomarker during early phase clinical trials. Citation Format: Nicole Schubert, Dennis Kirchhoff, Joumana Zeidan, Joanie Duchesne, Philip Boulais, Yuko Ishii. Phosphorylated extracellular signal-regulated kinase (pERK) activation in T effector cells as a target engagement biomarker for the DGKζ inhibitor BAY2965501 in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2116.

The phosphorylation of PHF5A by TrkA-ERK1/2-ABL1 cascade regulates centrosome separation

During interphase, the newly duplicated pairs of centrosomes are held together by a centrosome linker, and the centrosome separation needs the disruption of this linker to induce the duplicated centrosomes separating into two distinct microtubule organization centers. The mechanism of regulating centrosome separation is however poorly understood. Here, we demonstrated that the phosphorylation of PHF5A at Y36 by the TrkA-ERK1/2-ABL1 cascade plays a critical role in regulating centrosome separation. PHF5A, a well-characterized spliceosome component, is enriched in the centrosome. The pY36-PHF5A promotes the interaction between CEP250 and Nek2A in a spliceosomal-independent manner, which leads to premature centrosome separation. Furthermore, the unmatured centrosome remodels the microtubule and subsequently regulates cell proliferation and migration. Importantly, we found that the phosphorylation cascade of TrkA-ERK1/2-ABL1-PHF5A is hyper-regulated in medulloblastoma. The inhibition of this cascade can induce senescence and restrict the proliferation of medulloblastoma. Our findings on this phosphorylation cascade in regulating centrosome separation could provide a series of potential targets for restricting the progress of medulloblastoma.

Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression

Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes.

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.

The ring saga: looking back at the discovery of γ-tubulin and γ-tubulin ring complexes

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.

StarD7 deficiency hinders cell motility through p-ERK1/2/Cx43 reduction.

StarD7 belongs to START protein family involved in lipid traffic, metabolism, and signaling events. Its precursor, StarD7.I which is important for mitochondrial homeostasis, is processed to the StarD7.II isoform that lacks the mitochondrial targeting sequence and is mainly released to the cytosol. StarD7 knockdown interferes with cell migration by an unknown mechanism. Here, we demonstrate that StarD7 silencing decreased connexin 43 (Cx43), integrin β1, and p-ERK1/2 expression in the non-tumoral migratory HTR-8/SVneo cells. StarD7-deficient cells exhibited Golgi disruption and reduced competence to reorient the microtubule-organizing center. The migratory capacity of StarD7-silenced cells was reestablished when Cx43 level was resettled, while p-ERK1/2 expression remained low. Importantly, ectopic expression of the StarD7.II isoform not only restored cell migration but also ERK1/2, Cx43, and integrin β1 expression. Thus, StarD7 is implicated in cell migration through an ERK1/2/Cx43 dependent mechanism but independent of the StarD7.I function in the mitochondria.

Divergent Contribution of the Golgi Apparatus to Microtubule Organization in Related Cell Lines.

Membrane trafficking in interphase animal cells is accomplished mostly along the microtubules. Microtubules are often organized radially by the microtubule-organizing center to coordinate intracellular transport. Along with the centrosome, the Golgi often serves as a microtubule-organizing center, capable of nucleating and retaining microtubules. Recent studies revealed the role of a special subset of Golgi-derived microtubules, which facilitates vesicular traffic from this central transport hub of the cell. However, proteins essential for microtubule organization onto the Golgi might be differentially expressed in different cell lines, while many potential participants remain undiscovered. In the current work, we analyzed the involvement of the Golgi complex in microtubule organization in related cell lines. We studied two cell lines, both originating from green monkey renal epithelium, and found that they relied either on the centrosome or on the Golgi as a main microtubule-organizing center. We demonstrated that the difference in their Golgi microtubule-organizing activity was not associated with the well-studied proteins, such as CAMSAP3, CLASP2, GCC185, and GMAP210, but revealed several potential candidates involved in this process.

Mitotic progression and dual spindle formation caused by spindle association of de novo-formed microtubule-organizing centers in parthenogenetic embryos of Drosophila ananassae.

Facultative parthenogenesis occurs in many animal species that typically undergo sexual reproduction. In Drosophila, such development from unfertilized eggs involves diploidization after completion of meiosis, but the exact mechanism remains unclear. Here we used a laboratory stock of Drosophila ananassae that has been maintained parthenogenetically to cytologically examine the initial events of parthenogenesis. Specifically, we determined whether the requirements for centrosomes and diploidization that are essential for developmental success can be overcome. As a primal deviation from sexually reproducing (i.e. sexual) strains of the same species, free asters emerged from the de novo formation of centrosome-like structures in the cytosol of unfertilized eggs. Those microtubule-organizing centers had distinct roles in the earliest cycles of parthenogenetic embryos with respect to mitotic progression and arrangement of mitotic spindles. In the first cycle, an anastral bipolar spindle self-assembled around a haploid set of replicated chromosomes. Participation of at least one microtubule-organizing center in the spindle was necessary for mitotic progression into anaphase. In particular, the first mitosis involving a monastral bipolar spindle resulted in haploid daughter nuclei, one of which was associated with a microtubule-organizing center whereas the other was not. Remarkably, in the following cycle, biastral and anastral bipolar spindles formed that were frequently arranged in tandem by sharing an aster with bidirectional connections at their central poles. We propose that, for diploidization of haploid nuclei, unfertilized parthenogenetic embryos utilize dual spindles during the second mitosis, as occurs for the first mitosis in normal fertilized eggs.

Azoxystrobin exposure impairs meiotic maturation by disturbing spindle formation in mouse oocytes.

Fungicides are a type of pesticide used to protect plants and crops from pathogenic fungi. Azoxystrobin (AZO), a natural methoxyacrylate derived from strobilurin, is one of the most widely used fungicides in agriculture. AZO exerts its fungicidal activity by inhibiting mitochondrial respiration, but its cytotoxicity to mammalian oocytes has not been studied. In this study, we investigated the effect of AZO exposure on mouse oocyte maturation to elucidate the underlying mechanisms of its possible reproductive toxicity. We found that AZO exposure disturbed meiotic maturation by impairing spindle formation and chromosome alignment, which was associated with decreased microtubule organizing center (MTOC) integrity. Moreover, AZO exposure induced abnormal mitochondrial distribution and increased oxidative stress. The AZO-induced toxicity to oocytes was relieved by melatonin supplementation during meiotic maturation. Therefore, our results suggest that AZO exposure impairs oocyte maturation not only by increasing oxidative stress and mitochondrial dysfunction, but also by decreasing MTOC integrity and subsequent spindle formation and chromosome alignment.

Histone H1 – structure and function

Linker histones (H1) are proteins found in the nuclei of the vast majority of Eucaryota, playing important roles in their life and development. H1 takes part in processes such as chromatin condensation, transcriptional regulation of gene expression, apoptosis induction and many more. Despite its common presence and essential function, many questions remain unanswered. Experiments conducted to date don’t provide unambiguous information on such crucial issues as e.g. the way linker histones bind to nucleosome. There is also much surprising information about H1 participating in physiological processes not connected directly to its widely known function – e.g. providing a microtubule organization center in plants or contributing to the defense against pathogens in fish. The objective of the present work is to provide insights into many aspects of linker histone structure and function, collect and systematize current knowledge and to outline questions worth answering in the future.

PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) are required for microtubule polarity in Caenorhabditis elegans dendrites.

The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.

Microtubule organization and cell geometry.

A characteristic feature of nondividing animal cells is the radial organization of microtubules (MTs), emanating from a single microtubule organizing center (MTOC). As generically these cells are not spherically symmetric, this raises the question of the influence of cell geometry on the orientational distribution of microtubules. We present a systematic study of this question in a simplified setting where MTs are nucleated from a single fixed MTOC in the center of an elliptical cell geometry. Within this context we introduce four models of increasing complexity, each one introducing additional mechanisms that govern the interaction of the MTs with the cell boundary. In order, we consider the cases: MTs that can bind to the boundary with a fixed mean residence time (M0), force-producing MTs that can slide on the boundary towards the cell poles (MS), MTs that interact with a generic polarity factor that is transported and deposited at the boundary, and which in turn stabilizes the MTs at the boundary (MP), and a final model in which both sliding and stabilization by polarity factors is taken into account (MSP). In the baseline model (M0), the exponential length distribution of MTs causes most of the interactions at the cell boundary to occur along the shorter transverse direction in the cell, leading to transverse biaxial order. MT sliding (MS) is able to reorient the main axis of this biaxial order along the longitudinal axis. The polarization mechanism introduced in MP and MSP overrules the geometric bias towards bipolar order observed in M0 and MS, and allows the establishment of unipolar order either along the short (MP) or the long cell axis (MSP). The behavior of the latter two models can be qualitatively reproduced by a very simple toy model with discrete MT orientations.

Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells.

Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. Here, we demonstrate that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, we show that human Parafibromin rescues the ectopic NSC phenotype in Drosophila hyx mutant brains. We have also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. Our data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.

Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells.

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.

Emerging roles of centrosome cohesion.

The centrosome, consisting of centrioles and the associated pericentriolar material, is the main microtubule-organizing centre (MTOC) in animal cells. During most of interphase, the two centrosomes of a cell are joined together by centrosome cohesion into one MTOC. The most dominant element of centrosome cohesion is the centrosome linker, an interdigitating, fibrous network formed by the protein C-Nap1 anchoring a number of coiled-coil proteins including rootletin to the proximal end of centrioles. Alternatively, centrosomes can be kept together by the action of the minus end directed kinesin motor protein KIFC3 that works on interdigitating microtubules organized by both centrosomes and probably by the actin network. Although cells connect the two interphase centrosomes by several mechanisms into one MTOC, the general importance of centrosome cohesion, particularly for an organism, is still largely unclear. In this article, we review the functions of the centrosome linker and discuss how centrosome cohesion defects can lead to diseases.

Effect of Direct Current Electric Fields on Cone Like Retinal Photoreceptor Cells.

Studies show that electric fields are used as therapy during nerve and tissue injuries along with trans-retinal stimulation. However, cellular and molecular changes induced by such treatments remain largely unknown especially in retinal photoreceptor cells. In vitro studies show that direct current electric fields (dcEF) were known to influence cell division, polarity, shape, and motility. Here we could characterize for the first time the reactions of 661W, a retinal cone photoreceptor especially regarding organelle polarization, membrane polarization of mitochondria, O2 consumption, ATP/ADP ratio and gene expression. The 661W cells were stimulated with a constant dcEF of field strength 5 V/cm during 30 min or 5 h depending on the parameters studied. In response to dcEF, the cells aligned perpendicular to the field by forming a leading edge with extended membrane protrusions towards the cathode. Using immunofluorescence and live cell imaging, we show that the cell membrane depolarized at the cathodal side. The microtubules spread into the direction of migration. Also, the microtubule organization center re-oriented into this direction. Concomitantly with the microtubules, actin filaments reorganized in an asymmetrical fashion mainly at the cathodal side. The Golgi apparatus, which is involved in many steps of actin synthesis, moved to the cathodal side. In the last 2 h of the 5 h experiment, microtubules positioned themselves at the rear (anodal side), like the nucleus. The averaged displacement of the whole cells under dcEF was 155% of control for 3 V/cm and 235% for 5 V/cm. The average speed increased by 142% and 243% respectively. Inside the cells mitochondria moved to the cathodal side, where the energy consuming producing processes take place. In this line, we measured an increase in ATP production and O2 consumption. Mitochondrial calcium was found more on the anodal side, at the site of the nucleus with its calcium delivering endoplasmic reticulum. In addition, oxymetry studies reveal an increased ATP synthesis by 115.2% and oxygen consumption by 113.3% 3 h after dcEF stimulation. An analysis of differentially expressed genes by RNA sequencing revealed an upregulation of genes involved in cellular movement, cell to cell and intracellular signaling, molecular transport, assembly and organization. The mechanisms found can enhance our understanding regarding the beneficial effects of EF treatment in retinal diseases.

Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes

The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite’s nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and −4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo.

ABSTRACTIn embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

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.