Function Of Microtubule Organizing Centers Research Articles

Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly.

As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. Here, we investigate the post-transcriptional regulation of Plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. Our data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, we find that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. Our data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.

Sub-centrosomal mapping identifies augmin-\u03b3TuRC as part of a centriole-stabilizing scaffold

Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease.

Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia

During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C.elegans. Using tissue-specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis, while SPD-2/CEP192 and PCMD-1, which are essential in the one-cell embryo, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C.elegans,1-3 we find evidence for "centriole-less PCM" at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules toward the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

Centriole-Less Pericentriolar Material Serves as a Microtubule Organizing Center at the Base of C. elegans Sensory Cilia

During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis while SPD-2/CEP192 and PCMD-1, which are essential in the zygote, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans , we find evidence for “centriole-less PCM” at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules towards the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator [[EQUATION]] -TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome.

The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers, little is known about how the centrosome is inactivated as an MTOC. Analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of partially overlapping territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is mechanically ruptured by cortical pulling forces, ultimately inactivating MTOC function at the centrosome.

MTOC Organization and Competition During Neuron Differentiation.

Neurons are polarized cells with long branched axons and dendrites. Microtubule generation and organization machineries are crucial to grow and pattern these complex cellular extensions. Microtubule organizing centers (MTOCs) concentrate the molecular machinery for templating microtubules, stabilizing the nascent polymer, and organizing the resultant microtubules into higher-order structures. MTOC formation and function are well described at the centrosome, in the spindle, and at interphase Golgi; we review these studies and then describe recent results about how the machineries acting at these classic MTOCs are repurposed in the postmitotic neuron for axon and dendrite differentiation. We further discuss a constant tug-of-war interplay between different MTOC activities in the cell and how this process can be used as a substrate for transcription factor-mediated diversification of neuron types.

SPD-2/CEP192 and CDK Are Limiting for Microtubule-Organizing Center Function at the Centrosome

The centrosome acts as the microtubule-organizing center (MTOC) during mitosis in animal cells. Microtubules are nucleated and anchored by γ-tubulin ring complexes (γ-TuRCs) embedded within the centrosome's pericentriolar material (PCM). The PCM is required for the localization of γ-TuRCs, and both are steadily recruited to the centrosome, culminating in a peak in MTOC function in metaphase. In differentiated cells, the centrosome is often attenuated as an MTOC and MTOC function is reassigned to non-centrosomal sites such as the apical membrane in epithelial cells, the nuclear envelope in skeletal muscle, and down the lengths of axons and dendrites in neurons. Hyperactive MTOC function at the centrosome is associated with epithelial cancers and with invasive behavior in tumor cells. Little is known about the mechanisms that limit MTOC activation at the centrosome. Here, we find that MTOC function at the centrosome is completely inactivated during cell differentiation in C. elegans embryonic intestinal cells and MTOC function is reassigned to the apical membrane. In cells that divide after differentiation, the cellular MTOC state switches between the membrane and the centrosome. Using cell fusion experiments in live embryos, we find that the centrosome MTOC state is dominant and that the inactive MTOC state of the centrosome is malleable; fusion of a mitotic cell to a differentiated or interphase cell results in rapid reactivation of the centrosome MTOC. We show that conversion of MTOC state involves the conserved centrosome protein SPD-2/CEP192 and CDK activity from the mitotic cell.

Depletion of pericentrin in mouse oocytes disrupts microtubule organizing center function and meiotic spindle organization.

Accurate chromosome segregation is dependent on the formation and stability of the microtubule spindle apparatus. Meiotic spindle assembly in oocytes differs from the process used during mitosis, and is regulated by unique microtubule organizing centers (MTOCs) that lack centrioles. To gain insight into the molecular composition and function of acentriolar MTOCs in mouse oocytes, we assessed the role of a key MTOC-associated protein, pericentrin (PCNT). In somatic cells, pericentrin functions as a scaffold that binds specific proteins at MTOCs, including γ-tubulin, which is necessary for microtubule nucleation. Pericentrin is expressed in oocytes, but the conservation of its function is not known. Pericentrin localizes specifically to MTOCs during prophase-I arrest in mouse oocytes recovered from pre-ovulatory ovarian follicles, and remains associated with MTOCs at spindle poles during metaphase-I and -II. To test function, specific siRNAs were used to knock down Pcnt transcripts in mouse oocytes. Efficient protein depletion was confirmed by Western blot as well as immunofluorescence analysis. Notably, meiotic spindle structure and chromosome alignment were disrupted in Pcnt-depleted oocytes. Disorganized spindle structures with reduced microtubule density and misaligned chromosomes were observed in the majority of these oocytes (∼ 70%). In addition, γ-tubulin localization to MTOCs was significantly reduced and microtubule regrowth, following cold treatment, was delayed in Pcnt-depleted oocytes. Thus, pericentrin is a key functional component of the unique acentriolar MTOCs of mouse oocytes, and plays an important role in regulating meiotic spindle assembly and/or stability.

Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3(Alp6).

In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

Procedure for Bovine ICSI, not Sperm Freeze-drying, Impairs the Function of the Microtubule-organizing Center

This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.

Aurora A Kinase-Coated Beads Function as Microtubule-Organizing Centers and Enhance RanGTP-Induced Spindle Assembly

The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts . Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear . Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads , AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.

CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation.

The mammalian centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM). The architecture and composition of the centrosome, especially the PCM, changes during the cell cycle. Recently, a subset of PCM proteins have been shown to be arranged in a tubular conformation with an open and a closed end within the centrosome. The presence of such a specific configuration can be used as a landmark for mapping proteins in both a spatial and a temporal fashion. Such mapping studies can provide information about centrosome organization, protein dynamics, protein-protein interactions as well as protein function. In this study, the centrosomal proteins CEP110 and ninein were mapped in relationship to the tubular configuration. Both proteins were found to exhibit a similar distribution pattern. In the mother centrosome, they were found at both ends of the centrosome tube, including the site of centrosome duplication. However, in the daughter centrosome they were present only at the closed end. At the closed end of the mother and daughter centrosome tube, both CEP110 and ninein co-localized with the centriolar protein CEP250/c-Nap1, which confirms ninein's centriole association and places CEP110 in association with this structure. Importantly, the appearance of CEP110 and ninein at the open end of the daughter centrosome occurred during the telophase-G1 transition of the next cell cycle, concomitant with the maturation of the daughter centrosome into a mother centrosome. Microinjection of antibodies against either CEP110 or ninein into metaphase HeLa cells disrupted the reformation of the tubular conformation of proteins within the centrosome following cell division and consequently led to dispersal of centrosomal material throughout the cytosol. Further, microinjection of antibodies to either CEP110 or ninein into metaphase PtK2 cells not only disrupted the tubular configuration within the centrosome but also affected the centrosome's ability to function as a microtubule organizing center (MTOC). This MTOC function was also disrupted when the antibodies were injected into postmitotic cells. Taken together, our results indicate that: (1) a population of CEP110 and ninein is located in a specific domain within the centrosome, which corresponds to the open end of the centrosome tube and is the site of protein addition associated with maturation of a daughter centrosome into a mother centrosome; and (2) the addition of CEP110 and ninein are essential for the reformation of specific aspects of the interphase centrosome architecture following mitosis as well as being required for the centrosome to function as a MTOC.

Centrosome precursors in the acentriolar mouse oocyte

Centrioles disappear from the mouse oocyte during early oogenesis. However, it has been known for some time that multiple structures known as microtubule organizing centers (MTOC) form the spindle poles during meiosis as well as the mitotic poles during early cleavage. The objective of this study was to identify and describe the structures which exist prior to the appearance of the multiple MTOC associated with meiotic division. Reported here for the first time is a description of the unique structures which exist before the onset of oocyte maturation, their location, and microtubule (MT) nucleating ability. Correlative confocal, immuno-, and electron microscopic studies of mouse oocytes released from ovaries directly into 2% paraformaldehyde (time zero) show two large gamma-tubulin-positive structures in the cortex, averaging 10 microm in diameter. The present work is the first to demonstrate that although these structures contain gamma-tubulin, they do not resemble MTOC morphologically nor do they appear to nucleate MT. They are termed here multivesicular aggregates (MVA), and ultrastructural analysis reveals that they contain a variety of vesicular structures including many ring structures of approximately 25 nm. At the onset of maturation, these two MVA migrate toward the GV breaking into smaller units, only some of which mature into MTOC and nucleate MT. These correlative microscopic studies support the conclusion that MVA are centrosomal precursors, but with a unique ultrastructure. The ultrastructural organization of MVA may explain the cryptic function of MTOC in the prematuration environment of the dictyate oocyte.

The microtubule organizing centers of Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe ( S. pombe ) is assuming a higher profile in the study of diverse aspects of microtubule function during mitosis and in cellular morphogenesis. The study of S. pombe makes a major contribution to the understanding of how microtubules can control morphogenesis. The radical differences in the cell cycle organization, morphology, and great evolutionary distance that distinguish S. pombe from the most intensively studied model eukaryote, Saccharomyces cerevisiae ( S. cerevisiae ) , are discussed in the chapter. The formation of discrete microtubule organizing centers (MTOC) at distinct stages of the life cycle offers an attractive system for the study of de novo MTOC formation. The lack of any clear S. cerevisiae homologs for two of the handful of essential components of the S. pombe spindle pole body (SPB) that are identified suggests that S. pombe may reveal much about the degree of conservation of MTOC function between species. There are three distinct MTOC in S. pombe: (1) the SPB, which is present throughout the entire life cycle, (2) the equatorial MTOC (EMTOC), which forms at the end of mitosis at the site of cell division, and (3) the tip-associated MTOC (TAM), which forms at the cell tip during mating. Thus, the system offers many insights into the molecular basis of microtubule nucleation and lends itself to the study of de novo MTOC formation.

Essential role for gamma -tubulin in the acentriolar female meiotic spindle of Drosophila

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.

The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.