Non-centrosomal Microtubule-organizing Centers Research Articles

Ninein domains required for its localization, association with partners dynein and ensconsin, and microtubule organization.

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize microtubules at non-centrosomal microtubule-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates microtubule assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.

A distinct isoform of Msp300/Nesprin organizes the perinuclear microtubule organizing center in adipose cells.

In many cell types, disparate non-centrosomal microtubule-organizing centers (ncMTOCs) replace functional centrosomes and serve the unique needs of the cell types in which they are formed. In Drosophila fat body cells (adipocytes), an ncMTOC is organized on the nuclear surface. This perinuclear ncMTOC is anchored by Msp300, encoded by one of two Nesprin-encoding genes in Drosophila. Msp300 and the spectraplakin short stop (shot) are co-dependent for localization to the nuclear envelope to generate the ncMTOC where they recruit the microtubule minus-end stabilizer Patronin (CAMSAP). The fat body perinuclear ncMTOC requires Patronin, Ninein, and Msps (ortholog of ch-TOG), but does not require γ-tubulin for MT assembly. The Msp300 gene is complex, encoding at least eleven isoforms. Here we show that two Msp300 isoforms, Msp300-PE and -PG, are required and only one, Msp300-PE, appears sufficient for generation of the ncMTOC. Loss of Msp300-PE,-PG retains the presence of the other isoforms at the nuclear surface, indicating that they are not sufficient to generate the ncMTOC. Loss of Msp300-PE,-PG results in severe loss of localization of shot and Patronin, and disruption of the MT array. This results in nuclear mispositioning and loss of endosomal trafficking. Msp300-PE has an unusual domain structure including a lack of a KASH domain and very few spectrin repeats and appears therefore to have a highly derived function suited to generating an ncMTOC on the nuclear surface.

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 Reduction in Microtubule Arrays Caused by the Dysplasia of the Non-Centrosomal Microtubule-Organizing Center Leads to a Malformed Organ of Corti in the Cx26-Null Mouse.

Mutations in the GJB2 gene account for approximately 20–50% of all non-syndromic hereditary deafness cases. The malformed organ of Corti (OC) was observed in different Cx26-null mouse models, which was mainly caused by the developmental arrest of pillar cells (PCs). However, the mechanism of developmental abnormalities in PCs caused by Cx26 deletion is still unclear. In this study, the ultrastructure of PCs at different postnatal days was observed in Cx26-null mice. Knockout of cochlear Cx26 led to the malformed assembly of non-centrosomal microtubule-organizing centers (MTOCs) far from the centrosome rather than near the centrosome. Additionally, the microtubule (MT) arrays emitted by abnormal non-centrosomal MTOCs were significantly reduced. In addition, we found that the protein expression of calmodulin-regulated, spectrin-associated protein2 (camsap2), a microtubule minus-end targeting protein associated with the organization of non-centrosomal MTs, was decreased in juvenile PCs in the Cx26-null group. Our results indicated that the malformation of non-centrosomal MTOCs in cochlear PCs might lead to the corresponding MTs’ failure to be captured and anchored in Cx26-null mice, which results in the deformity of OC. Additionally, this abnormal developmental process might be correlated with the reduced expression of camsap2 caused by Cx26 deletion in the early developmental stage.

Myogenin controls via AKAP6 non-centrosomal microtubule-organizing center formation at the nuclear envelope

Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6β and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.

The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control.

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) have been described in many cell types. The functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans, additional MTOCs have been discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates the activities of the SPB outer plaque and the sMTOC. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with γ-TuRC receptors.

Proximity Labeling at Non-Centrosomal Microtubule-Organizing Centers Reveals VAB-10B and WDR-62 as Distinct Microtubule Regulators

Microtubules are polarized intracellular polymers that play key roles in the cell including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome, however most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based proximity labeling approach TurboID for use in C. elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. Depletion of VAB-10B resulted in disorganized microtubules and delayed localization of a microtubule nucleation complex γ-TuRC while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, our study expands the basic cell biological roles of this important disease protein. Together, our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes.

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.

Alp7-Mto1 and Alp14 synergize to promote interphase microtubule regrowth from the nuclear envelope.

Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE.

Centrosomal and Non-Centrosomal Microtubule-Organizing Centers (MTOCs) in Drosophila melanogaster.

The centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in particular cell types and at specific stages during Drosophila development. The centrosome is not required zygotically for mitosis or to achieve full animal development. Nevertheless, centrosomes are essential maternally during cleavage cycles in the early embryo, for male meiotic divisions, for efficient division of epithelial cells in the imaginal wing disc, and for cilium/flagellum assembly in sensory neurons and spermatozoa. Importantly, asymmetric and polarized division of stem cells is regulated by centrosomes and by the asymmetric regulation of their microtubule (MT) assembly activity. More recently, the components and functions of a variety of non-centrosomal microtubule-organizing centers (ncMTOCs) have begun to be elucidated. Throughout Drosophila development, a wide variety of unique ncMTOCs form in epithelial and non-epithelial cell types at an assortment of subcellular locations. Some of these cell types also utilize the centrosomal MTOC, while others rely exclusively on ncMTOCs. The impressive variety of ncMTOCs being discovered provides novel insight into the diverse functions of MTOCs in cells and tissues. This review highlights our current knowledge of the composition, assembly, and functional roles of centrosomal and non-centrosomal MTOCs in Drosophila.

PEDF regulates plasticity of a novel lipid-MTOC axis in prostate cancer-associated fibroblasts.

Prostate tumors make metabolic adaptations to ensure adequate energy and amplify cell cycle regulators, such as centrosomes, to sustain their proliferative capacity. It is not known whether cancer-associated fibroblasts (CAFs) undergo metabolic re-programming. We postulated that CAFs augment lipid storage and amplify centrosomal or non-centrosomal microtubule-organizing centers (MTOCs) through a pigment epithelium-derived factor (PEDF)-dependent lipid-MTOC signaling axis. Primary human normal prostate fibroblasts (NFs) and CAFs were evaluated for lipid content, triacylglycerol-regulating proteins, MTOC number and distribution. CAFs were found to store more neutral lipids than NFs. Adipose triglyceride lipase (ATGL) and PEDF were strongly expressed in NFs, whereas CAFs had minimal to undetectable levels of PEDF or ATGL protein. At baseline, CAFs demonstrated MTOC amplification when compared to 1-2 perinuclear MTOCs consistently observed in NFs. Treatment with PEDF or blockade of lipogenesis suppressed lipid content and MTOC number. In summary, our data support that CAFs have acquired a tumor-like phenotype by re-programming lipid metabolism and amplifying MTOCs. Normalization of MTOCs by restoring PEDF or by blocking lipogenesis highlights a previously unrecognized plasticity in centrosomes, which is regulated through a new lipid-MTOC axis.This article has an associated First Person interview with the first author of the paper.

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization toexert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (γ-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.

The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila.

Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division.

Membrane repolarization is delayed in proximal tubules after ischemia-reperfusion: possible role of microtubule-organizing centers.

We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1 and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308-1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure.