Kinetics and intermediates of marginal band reformation: evidence for peripheral determinants of microtubule organization.

The microtubules of the mature erythrocyte of the chicken are confined to a band at the periphery. Whole-mount electron microscopy after extraction reveals that the number of microtubules in each cell is almost the same. All the microtubules can be depolymerized by incubation in the cold, and the marginal band can be quantitatively and qualitatively reformed by return to 39 degrees C. These properties allow the reformation of the marginal band to be treated as an in vivo microtubule assembly reaction. The kinetics of this reaction and the intermediates detected during reformation suggest a mechanism of microtubule organization that is distinct from that observed in other cell types. Apparently only one or two growing microtubule ends are available for assembly--assembly is only detected at the cell periphery, even at early times--and there is no evidence of the participation of a microtubule-organizing center.

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In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties.

The Journal of Cell Biology ◽

10.1083/jcb.118.4.813 ◽

1992 ◽

Vol 118 (4) ◽

pp. 813-830 ◽

Cited By ~ 42

Author(s):

J Tooze ◽

M Hollinshead

Keyword(s):

Hela Cells ◽

Brefeldin A ◽

Cell Types ◽

Morphological Evidence ◽

Detectable Effect ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Open Network ◽

Early Endosomes ◽

Organizing Center

We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA than the early endosomes. The morphological evidence suggests that all the tubular early endosomes in BFA-treated HeLa and AtT20 cells are linked together in a single reticulum. Consistent with this, incubations as short as 1-3 min with 10 or 20 mg/ml HRP in the medium result in the entire endosomal reticulum in most of the BFA-treated cells being filled with HRP reaction product.(ABSTRACT TRUNCATED AT 400 WORDS)

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Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e09-05-0415 ◽

2009 ◽

Vol 20 (22) ◽

pp. 4816-4825 ◽

Cited By ~ 24

Author(s):

Stefan Koch ◽

Christopher T. Capaldo ◽

Stanislav Samarin ◽

Porfirio Nava ◽

Irmgard Neumaier ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Leading Edge ◽

Cell Types ◽

Intestinal Epithelial ◽

Microtubule Organizing Center ◽

Organizing Center ◽

Interfering Rna

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.

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Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

The Journal of Cell Biology ◽

10.1083/jcb.83.3.623 ◽

1979 ◽

Vol 83 (3) ◽

pp. 623-632 ◽

Cited By ~ 28

Author(s):

M Schliwa ◽

U Euteneuer ◽

W Herzog ◽

K Weber

Keyword(s):

Complete Removal ◽

Cell System ◽

The State ◽

Microtubule Assembly ◽

Microtubule Organizing Center ◽

Functional Changes ◽

Organizing Center ◽

Pigment Distribution ◽

And Function ◽

In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

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

Journal of Cell Science ◽

10.1242/jcs.115.9.1825 ◽

2002 ◽

Vol 115 (9) ◽

pp. 1825-1835 ◽

Cited By ~ 1

Author(s):

Young Y. Ou ◽

Gary J. Mack ◽

Meifeng Zhang ◽

Jerome B. Rattner

Keyword(s):

Cell Cycle ◽

Protein Interactions ◽

Protein Function ◽

Similar Distribution ◽

Microtubule Organizing Center ◽

Specific Domain ◽

The Reformation ◽

Mother And Daughter ◽

Organizing Center ◽

Pericentriolar Material

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.

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The centriolar complex isolated from starfish spermatozoa

Journal of Cell Science ◽

10.1242/jcs.49.1.33 ◽

1981 ◽

Vol 49 (1) ◽

pp. 33-49 ◽

Cited By ~ 1

Author(s):

R. Kuriyama ◽

H. Kanatani

Keyword(s):

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Microtubule Assembly ◽

High Concentration ◽

Microtubule Organization ◽

Sucrose Density Gradient Centrifugation ◽

The Difference ◽

Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

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Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0063 ◽

2010 ◽

Vol 21 (1) ◽

pp. 18-28 ◽

Cited By ~ 29

Author(s):

Claudia Lang ◽

Sandrine Grava ◽

Tineke van den Hoorn ◽

Rhonda Trimble ◽

Peter Philippsen ◽

...

Keyword(s):

Spindle Pole Body ◽

Hyphal Growth ◽

Spindle Pole ◽

Microtubule Organizing Center ◽

Microtubule Nucleation ◽

Cytoplasmic Microtubule ◽

Organizing Center ◽

Cytoplasmic Side ◽

Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

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

Journal of Cell Science ◽

10.1242/jcs.256537 ◽

2021 ◽

Author(s):

Xiaolei Gao ◽

Saturnino Herrero ◽

Valentin Wernet ◽

Sylvia Erhardt ◽

Oliver Valerius ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Cell Types ◽

Spindle Pole ◽

Receptor Protein ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Spindle Pole Bodies ◽

Organizing Center ◽

Ring Complex ◽

Core Components

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) were described in many cell types. Functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans additional MTOCs were 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 SPB outer plaque with sMTOC activities. 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 polo-like kinase as a regulator of MTOC activities and as a scaffolding unit through interaction with γ-tubulin ring complex receptors.

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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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Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922911117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19254-19265

Author(s):

Jianming Liu ◽

Zhan-Peng Huang ◽

Mao Nie ◽

Gang Wang ◽

William J. Silva ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Function ◽

Neuromuscular Junctions ◽

Linc Complex ◽

Muscle Dystrophy ◽

Microtubule Organizing Center ◽

Anchoring Protein ◽

Organizing Center ◽

Genetic Deletion

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy inmdxmice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

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Imaging of plasmacytoid dendritic cell interactions with T cells

Blood ◽

10.1182/blood-2008-02-139865 ◽

2009 ◽

Vol 113 (1) ◽

pp. 75-84 ◽

Cited By ~ 33

Author(s):

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

María López-Bravo ◽

Noa B. Martín-Cofreces ◽

Alix Scholer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Plasmacytoid Dendritic Cell ◽

Time Lapse ◽

Cell Interactions ◽

Type I ◽

Microtubule Organizing Center ◽

Immune Synapse ◽

Organizing Center

Abstract Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4+ T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-θ, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC–T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

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