Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs.

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.

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

Cells ◽

10.3390/cells7090121 ◽

2018 ◽

Vol 7 (9) ◽

pp. 121 ◽

Cited By ~ 18

Author(s):

Marisa Tillery ◽

Caitlyn Blake-Hedges ◽

Yiming Zheng ◽

Rebecca Buchwalter ◽

Timothy Megraw

Keyword(s):

Current Knowledge ◽

Cell Types ◽

Wing Disc ◽

Early Embryo ◽

Microtubule Organizing Center ◽

Drosophila Development ◽

Functional Roles ◽

Animal Development ◽

Microtubule Organizing Centers ◽

Organizing Center

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.

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Immunoprobe Localization by Correlative Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927600000337 ◽

2000 ◽

Vol 6 (3) ◽

pp. 195-201 ◽

Cited By ~ 2

Author(s):

Patricia G. Calarco

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Ultrastructural Level ◽

Correlative Microscopy ◽

Microtubule Organizing Center ◽

Large Size ◽

Organizing Center ◽

Gamma Tubulin ◽

High Level ◽

And Function

AbstractMammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

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A novel structural component of the Dictyostelium centrosome

Journal of Cell Science ◽

10.1242/jcs.109.13.3103 ◽

1996 ◽

Vol 109 (13) ◽

pp. 3103-3112 ◽

Cited By ~ 2

Author(s):

A. Kalt ◽

M. Schliwa

Keyword(s):

Mitotic Spindle ◽

Structural Component ◽

Myosin Ii ◽

Live Cells ◽

Microtubule Organizing Center ◽

Direct Role ◽

Cytoplasmic Microtubule ◽

Interphase Cells ◽

Organizing Center

The microtubule-organizing center of D. discoideum is a nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular mass component of the NAB which was identified by a monoclonal antibody raised against isolated nucleus/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350 kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it may also be found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatment with 2 M urea or 0.6 M KCl. The 350 kDa antigen was part of the thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350 kDa antigen is a structural component of the NAB corona which could be involved in its stabilization.

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The Role of the Centrosome in Development and Progression of Breast Cancer

Microscopy and Microanalysis ◽

10.1017/s1431927600028981 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 582-583

Author(s):

W. Lingle ◽

J. Salisbury ◽

S. Barrett ◽

V. Negron ◽

C. Whitehead

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Microtubule Organizing Center ◽

Daughter Cells ◽

Spindle Poles ◽

Sister Chromatids ◽

Close Proximity ◽

Interphase Cells ◽

Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

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A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

The Journal of Cell Biology ◽

10.1083/jcb.103.5.1863 ◽

1986 ◽

Vol 103 (5) ◽

pp. 1863-1872 ◽

Cited By ~ 25

Author(s):

P R Sager ◽

N L Rothfield ◽

J M Oliver ◽

R D Berlin

Keyword(s):

Mitotic Spindle ◽

Spindle Pole ◽

Early Prophase ◽

Microtubule Organizing Center ◽

Spindle Poles ◽

Interphase Cells ◽

Organizing Center ◽

Mitotic Spindle Pole ◽

Pole Component

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

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Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii

eLife ◽

10.7554/elife.56635 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Nicolò Tosetti ◽

Nicolas Dos Santos Pacheco ◽

Eloïse Bertiaux ◽

Bohumil Maco ◽

Lorène Bournonville ◽

...

Keyword(s):

Electron Microscopy ◽

Toxoplasma Gondii ◽

Polymer Fibers ◽

Microtubule Organizing Center ◽

Polar Ring ◽

Catastrophic Loss ◽

Organizing Center ◽

Infected Cells ◽

Essential Function ◽

Apical Complex

The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize the Toxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Parasites conditionally depleted in AC9 or AC10 replicate normally but are defective in microneme secretion and fail to invade and egress from infected cells. Electron microscopy revealed that the mature parasite mutants are conoidless, while U-ExM highlighted the disorganization of the SPMTs which likely results in the catastrophic loss of APR and conoid.

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The centriolar satellite protein Cfap53/Ccdc11 facilitates the formation of the first zygotic microtubule organizing center in the zebrafish embryo

10.1101/2020.11.18.388652 ◽

2020 ◽

Author(s):

Sven Willekers ◽

Federico Tessadori ◽

Babet van der Vaart ◽

Heiko Henning ◽

Riccardo Stucchi ◽

...

Keyword(s):

Cell Cycle ◽

Danio Rerio ◽

Mitotic Spindle ◽

Sperm Cell ◽

Zebrafish Embryo ◽

Animal Species ◽

Microtubule Organizing Center ◽

Microtubule Network ◽

Cell Divisions ◽

Organizing Center

AbstractIn embryos from most animal species a 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 mitotic spindle in the first and all subsequent cell divisions. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the first zygotic MTOC. Here we find that zebrafish (Danio rerio) embryos lacking maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle due to a failure in proper formation of the mitotic spindle. During the first cell cycle Cfap53 co-localizes with γ-tubulin and other centrosomal and centriolar satellite proteins to the very large MTOC. Furthermore, we find that γ-tubulin localization to the MTOC is impaired in the absence of Cfap53 or when the microtubule network is disrupted. Based on these results we propose a model in which maternal and paternal Cfap53 participates in the organization of the first zygotic MTOC of the embryo. Once the zygotic MTOC is formed, Cfap53 is dispensable for MTOC formation and integrity in subsequent cell divisions.

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Immunoprobe Localization by Correlative Microscopy

Microscopy and Microanalysis ◽

10.1007/s100059910021 ◽

2000 ◽

Vol 6 (3) ◽

pp. 195-201

Author(s):

Patricia G. Calarco

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Ultrastructural Level ◽

Correlative Microscopy ◽

Microtubule Organizing Center ◽

Large Size ◽

Organizing Center ◽

Gamma Tubulin ◽

High Level ◽

And Function

Abstract Mammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

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Monoplastidy in spermatogenesis of Lycopodium obscurum

Canadian Journal of Botany ◽

10.1139/b94-177 ◽

1994 ◽

Vol 72 (10) ◽

pp. 1436-1444 ◽

Cited By ~ 9

Author(s):

Karen Sue Renzaglia ◽

Angel R. Maden ◽

Jeffrey G. Duckett ◽

Dean P. Whittier

Keyword(s):

Endoplasmic Reticulum ◽

Key Words ◽

Mother Cell ◽

Annulate Lamellae ◽

Microtubule Organizing Center ◽

Microtubule Organizing Centers ◽

Cell Divisions ◽

Organizing Center ◽

Spindle Microtubules ◽

Spindle Development

Unlike Lycopodium laterale, which is polyplastidic during spermatogenesis, Lycopodium obscurum exhibits monoplastidy beginning in the early proliferative stages of antheridial development. Previous cell generations are polyplastidic and plastid fusion involving connective cylinders establishes the monoplastidic condition. Plastid and nuclear divisions are coordinated in L. obscurum with the plastids positioned at opposite poles prior to spindle development. Unlike monoplastidic cell divisions with morphogenetic plastid migration and polarity in other lycophytes, mosses, and hornworts, however, the spindles in L. obscurum do not originate from the plastid envelopes but from endoplasmic reticulum positioned against the plastid. In the final divisions, spindle microtubules emanate from structurally defined microtubule organizing centers that develop between the plastids and nucleus. Preceding the appearance of centrioles in the spermatid mother cell, the centrosomes comprise electron-dense granular matrices with associated vesicles and endoplasmic reticulum. Among archegoniate microtubule organizing centers, the discrete acentriolar centrosomes of Lycopodium most closely resemble the microtubule organizing centers in moss spore development and the polar organizer of liverwort mitosis. Key words: annulate lamellae, centrosome, Lycopodium, microtubule organizing center, monoplastidy, plastid dividing ring.

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

eLife ◽

10.7554/elife.47867 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

Author(s):

Jérémy Magescas ◽

Jenny C Zonka ◽

Jessica L Feldman

Keyword(s):

Cell Cycle ◽

Mitotic Spindle ◽

Outer Sphere ◽

Microtubule Organizing Center ◽

C Elegans ◽

Mitotic Kinases ◽

Pulling Forces ◽

Organizing Center ◽

Pericentriolar Material ◽

Step Mechanism

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.

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