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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 withmembers 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 colocalizationwas detected between MztA and the outer plaque receptor ApsB (fission yeastMto1). SeptalMTOCs 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. [ABSTRACT FROM AUTHOR]

During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plusends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-enddirected kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation. [ABSTRACT FROM AUTHOR]

Mouse oocytes lack canonical centrosomes and instead contain unique acentriolar microtubule-organizing centers (aMTOCs). To test the function of these distinct aMTOCs in meiotic spindle formation, pericentrin (Pcnt), an essential centrosome/MTOC protein, was knocked down exclusively in oocytes by using a transgenic RNAi approach. Here, we provide evidence that disruption of aMTOC function in oocytes promotes spindle instability and severe meiotic errors that lead to pronounced female subfertility. Pcnt-depleted oocytes from transgenic (Tg) mice were ovulated at the metaphase-II stage, but show significant chromosome misalignment, aneuploidy and premature sister chromatid separation. These defects were associated with loss of key Pcnt-interacting proteins (γ-tubulin, Nedd1 and Cep215) from meiotic spindle poles, altered spindle structure and chromosome–microtubule attachment errors. Live-cell imaging revealed disruptions in the dynamics of spindle assembly and organization, together with chromosome attachment and congression defects. Notably, spindle formation was dependent on Ran GTPase activity in Pcnt-deficient oocytes. Our findings establish that meiotic division is highly error-prone in the absence of Pcnt and disrupted aMTOCs, similar to what reportedly occurs in human oocytes. Moreover, these data underscore crucial differences between MTOC-dependent and -independent meiotic spindle assembly. [ABSTRACT FROM AUTHOR]

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes. [ABSTRACT FROM AUTHOR]

Microtubule-organizing centers such as the c-tubulin ring complex (γ-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast ctubulin small complex (γ-TuSC) of the γ-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast γ-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the γ-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human γ-TuSC proteins, we found that GCP3 assembles normally into the .2000 kDa fission yeast γ-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of γ-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast γ-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during γ-TuRC assembly. Our findings have broad importance for understanding the essential domains of γ-TuSC proteins in the γ-TuRC mechanism. [ABSTRACT FROM AUTHOR]

The function of microtubules depends on their arrangement into highly ordered arrays. Spatio-temporal control over the formation of new microtubules and regulation of their properties are central to the organization of these arrays. The nucleation of new microtubules requires γ-tubulin, an essential protein that assembles into multi-subunit complexes and is found in all eukaryotic organisms. However, the way in which γ-tubulin complexes are regulated and how this affects nucleation and, potentially, microtubule behavior, is poorly understood. γ-tubulin has been found in complexes of various sizes but several lines of evidence suggest that only large, ring-shaped complexes function as efficient microtubule nucleators. Human γ-tubulin ring complexes (γTuRCs) are composed of γ-tubulin and the γ-tubulin complex components (GCPs) 2, 3, 4, 5 and 6, which are members of a conserved protein family. Recent work has identified additional unrelated &3947;TuRC subunits, as well as a large number of more transient γTuRC interactors. In this Commentary, we discuss the regulation of γTuRC-dependent microtubule nucleation as a key mechanism of microtubule organization. Specifically, we focus on the regulatory roles of the γTuRC subunits and interactors and present an overview of other mechanisms that regulate γTuRC-dependent microtubule nucleation and organization. [ABSTRACT FROM AUTHOR]

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in viva fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation ΔkinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtubule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity that allows for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in Caenorhabditis elegans neurons, and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for correct microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron, however, use an additional pathway to nucleate microtubules at the tip of the dendrite, from the base of the cilium in ciliated neurons. Since inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule-organizing center at the dendrite tip ensures correct dendritic cargo transport., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)