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  • Articles  (17)
  • Cell Biology
  • Centrosome
  • Springer  (14)
  • Cell Press  (3)
  • Molecular Diversity Preservation International (MDPI)
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  • 1
    Publication Date: 2022-10-27
    Description: © The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Meaders, J. L., de Matos, S. N., & Burgess, D. R. A pushing mechanism for microtubule aster positioning in a large cell type. Cell Reports, 33(1), (2020): 108213, doi:10.1016/j.celrep.2020.108213.
    Description: After fertilization, microtubule (MT) sperm asters undergo long-range migration to accurately position pronuclei. Due to the large sizes of zygotes, the forces driving aster migration are considered to be from pulling on the astral MTs by dynein, with no significant contribution from pushing forces. Here, we re-investigate the forces responsible for sperm aster centration in sea urchin zygotes. Our quantifications of aster geometry and MT density preclude a pulling mechanism. Manipulation of aster radial lengths and growth rates, combined with quantitative tracking of aster migration dynamics, indicates that aster migration is equal to the length of rear aster radii, supporting a pushing model for centration. We find that dynein inhibition causes an increase in aster migration rates. Finally, ablation of rear astral MTs halts migration, whereas front and side ablations do not. Collectively, our data indicate that a pushing mechanism can drive the migration of asters in a large cell type.
    Description: We would like to thank Dr. Jesse Gatlin for sending us the Tau-mCherry fusion protein for imaging live MTs. We would also like to thank Dr. Timothy Mitchison, Dr. Christine Field, and Dr. James Pelletier for supplying us with CA4, p150-CC1, and EB1-GFP peptides, as well as for fruitful discussions. Finally, we would like to thank Dr. Charles Shuster and Leslie Toledo-Jacobo for constructive feedback when preparing the manuscript. We thank Bret Judson and the Boston College Imaging Core for infrastructure and support. This material is based upon work supported by NSF grant no. 124425 to D.R.B.
    Keywords: Dynein ; Aster ; Microtubule ; Centrosome ; Pronucleus ; Fertilization ; Aster position
    Repository Name: Woods Hole Open Access Server
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  • 2
    Publication Date: 2022-05-26
    Description: © The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hirst, W. G., Kiefer, C., Abdosamadi, M. K., Schäffer, E., & Reber, S. In Vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy. STAR Protocols, 1(3), (2020): 100177, doi:10.1016/j.xpro.2020.100177.
    Description: Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy.
    Description: We thank the AMBIO imaging facility (Charité, Berlin) and Nikon at MBL for imaging support. We thank all former and current members of the Reber lab for discussion and helpful advice, in particular Christoph Hentschel and Soma Zsoter for technical assistance. S.R. acknowledges funding by the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. C.K. thanks the Deutsche Forschungsgesellschaft (DFG, JA 2589/1-1). C.K. and M.A. thank Steve Simmert and Tobias Jachowski former and current members of the Schäffer lab.
    Keywords: Biophysics ; Cell Biology ; Microscopy
    Repository Name: Woods Hole Open Access Server
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  • 3
    Publication Date: 2022-05-26
    Description: © The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Geisterfer, Z. M., Oakey, J., & Gatlin, J. C. . Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography. STAR Protocols, 1(3), (2020): 100221, doi:10.1016/j.xpro.2020.100221.
    Description: Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).
    Description: This work was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant no. 2P20GM103432. It was also supported by additional funding provided by the NIGMS under grant no. R01GM113028, the NSF Faculty CAREER Program under award no. BBBE 1254608, Whitman Center fellowships at the Marine Biological Laboratory, and the Biomedical Scholars program of the Pew Charitable Trusts. We thank Drs. Aaron Groen and Tim Mitchison for their intellectual contributions and involvement in some of the pioneering experiments that set the foundation for this approach.
    Keywords: Biophysics ; Cell Biology ; Cell isolation ; Microscopy ; Model Organisms
    Repository Name: Woods Hole Open Access Server
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  • 4
    ISSN: 1432-041X
    Keywords: Key words α-Tubulin ; Centrosome ; Polar body formation ; Androgenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster.
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  • 5
    ISSN: 1423-0127
    Keywords: Kinase ; Mitosis ; Cell cycle ; Centrosome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous toDrosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, over-expression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.
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  • 6
    ISSN: 1615-6102
    Keywords: Centrosome ; Chemotaxis ; Microtubules ; Nucleus ; Pseudopod
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell locomotion. The centrosome is positioned between the leading edge pseudopod and the nucleus. This suggests that the polarized distribution of organelles in the cytoplasm is coupled spatially with structural and functional polarity in the cell cortex. To study cellular polarization with special interest in the roles of microtubules, we have analyzed the effects of microtubule-disrupting reagents and local laser irradiation on behaviors of both the nucleus and the centrosome in living amoebae ofPhysarum polycephalum. Physarum cells often have 2–3 pseudopods. One of the pseudopods keeps extending to become a stable leading edge while the rest retracts, a crucial step that reorients cells during locomotion. The nucleus, together with the centrosome, moves specifically toward the pseudopod that will become the leading edge. Disruption of microtubules with nocodazole randomizes positions of the nucleus, indicating the involvement of microtubules in the directional migration of the nucleus toward a specific pseudopod. The migration direction of the nucleus is reversed immediately after the UV laser is irradiated at regions between the nucleus and the future leading pseudopod. In contrast, irradiation at regions between the future tail and the nucleus does not affect nuclear migration. By immunofluorescence, we confirmed fragmentation of microtubules specifically in the irradiated region. These results suggest that the nucleus is pulled together with the centrosome toward the future leading-edge pseudopod in a microtubule-dependent manner. Microtubules seem to exert the pulling force generated in the cell cortex on the centrosome. They may serve as a mediator of shape changes initiated in the cell cortex to the organelle geometry in the endoplasm.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 196 (1997), S. 45-54 
    ISSN: 1615-6102
    Keywords: Allomyces macrogynus ; Centrosome ; Microtubule ; Microtubule-organizing centers ; Zoospores
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.
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  • 8
    ISSN: 1615-6102
    Keywords: Apical cell ; Centrin ; Centrosome ; Mictrotubule ; Mitosis ; Phaeophyceae ; Sphacelaria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Treatment of young thalli ofSphacelaria rigidula with 0.04 μg of nocodazole (Nz) per ml for up to 36 h affects microtubules (Mts) only slightly, but blocks a large number of mitotic cells in metaphase, without disruption of the metaphase plate. Higher concentrations of Nz (0.1 μg/ml) depolymerize interphase Mts. Only a few perinuclear and some short Mts resist and remain associated with the centrosomes. Fragmented Mts or groups of Mts sometimes remain in the apical dome. After treatment with 0.1 μg of Nz per ml, prometaphase cells are blocked at metaphase, while post-metaphase cells become binuclear, due to the failure of cytokinesis. With anticentrin immunofluorescence, a positive centrin signal is always observed in the centrosome area. Centrosome duplication is not affected by Nz, but separation is disturbed. After recovering for 2–4 h, most of the blocked metaphases proceed normally. In such cells duplicated centrosomes are seen in different stages of separation. In some cells independent aster-like microtubule configurations appear in the apical dome, occasionally displaying centrin at their centre. During recovery various configurations of bimitosis or multipolar mitosis were found. The multipolar spindles may share common centrosomes. Up to four centrosomes may accompany each nucleus. In some 24 h treated cells, as well as in cells recovering for 2 h, the centrin-positive structure is rod-like, extending in opposite directions from the usual position to the poles. Electron microscopical examination of thin sections revealed that the growth pattern of the apical cells is disrupted after Nz treatment. The observations show that: (a) the Mt cytoskeleton is involved in maintaining the polarity and growth pattern of apical cells, (b) mitosis is blocked by low concentrations of Nz without significant depolymerization of Mts, (c) the centrosome cycle is independent of the nuclear cycle, (d) centrosome separation and differentiation are disturbed by Nz treatment, (e) during recovery from Nz treatment, centrosomal material that may have separated from the centrosomes, as well as Mt fragments that resisted depolymerization, may operate as Mt nucleation centres.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 178 (1994), S. 97-110 
    ISSN: 1615-6102
    Keywords: Centrioles ; Centrosome ; Fertilization ; Fucus distichus ; Microtubules ; Mitosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Processes of fertilization and zygote development inFucus distichus were studied by indirect immunofluorescence microscopy using anti-β tubulin antibody and electron microscopy. Just after plasmogamy, sperm aster formation occurs during migration of a sperm nucleus toward an egg nucleus at the center of cytoplasm. Only sparse microtubules (MTs) exist around the egg nucleus. The sperm aster can be observed till karyogamy, but afterwards vanishes. Accompanying sperm aster formation, cortical MTs which are reticulately arranged develop further in the zygotes. In 4 h-old zygotes, characteristic structures which are composed of fine granular masses and consist of intermixed dense and lighter staining areas appear around the nucleus. These structures cannot be detected with anti-β tubulin immunofluorescence microscopy. The two centrioles derived from the sperm separate and migrate to both poles. In 4 h-and 8 h-old zygotes, there are no defined MT foci around the zygote nucleus and MTs radiate from the circumference of it. In 12 h-old zygotes, each centriole has migrated to the poles and derivative centrioles are generated. The fine granular masses also migrate to both poles and finally disappear accompanying the appearance of numerous MTs radiating from the poles. Therefore, two distinct MT foci appear from 12 h onwards. Progressive stages of nuclear division were also examined with electron and immunofluorescence microscopy in 16 h-old zygotes. The sperm chloroplast with an eyespot and the sperm mitochondria with an intercristal tubular structure, which are distinctive from those of egg, can be detected after plasmogamy and karyogamy. The sperm chloroplast is still present in 16 h-old zygotes.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 189 (1995), S. 203-215 
    ISSN: 1615-6102
    Keywords: Centrin ; Calmodulin ; EF-hand ; Centrosome ; Chlamydomonas ; Central helix
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Centrin is a unique member of the EF-hand superfamily that is found in calcium-modulated contractile fibers associated with the centrosomes in a wide variety of higher and lower eukaryotes. InChlamydomonas reinhardtii centrin is encoded by the genevfl 2. We previously showed that thevfl 2–220 mutation is a glutamic acid to lysine change at position 101 that results in the loss of the centrin-containing fibrous structures. Here we describe several phenotypic revertants of thevfl 2–220 mutation that carry second site suppressors at amino acid position 96 or 104. Even though these new mutations are able to suppress the variable flagellar number phenotype, they are not wild type at the ultrastractural level but instead show specific defects in their distal striated fibers and transition-region stellate fibers. The nucleus-basal body connectors are, however, functional, suggesting that the reversion of the variable flagellar number phenotype occurs through restoration of proper connections of the basal body apparatus to the nucleus, as has been previously proposed. Positions 96, 101, and 104 lie within centrin's central helix, indicating that, as is the case for calmodulin, this part of the protein is of particular importance to its function.
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