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ABSTRACT: After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)-III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III-mediated fission of large membrane structures.
Science 02/2011; 331(6024):1616-20. · 31.20 Impact Factor
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Sider Penkov,
Fanny Mende,
Vyacheslav Zagoriy,
Cihan Erkut,
René Martin,
Ulrike Pässler,
Kai Schuhmann,
Dominik Schwudke,
Margit Gruner, Jana Mäntler,
Thomas Reichert-Müller,
Andrej Shevchenko,
Hans-Joachim Knölker,
Teymuras V Kurzchalia
Angewandte Chemie International Edition 11/2010; 49(49):9430-5. · 13.45 Impact Factor
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ABSTRACT: HeLa cells are widely used as a model system to study cell division. The last step of cell division, abscission, occurs at an about 1 μm wide intercellular bridge that connects the post-mitotic sister cells. Abscission often occurs long after ingression of the cleavage furrow, and no efficient methods to synchronize cells to this stage are available. Here, we have developed a correlative fluorescence time-lapse imaging and electron microscopic approach using Aclar sheets with engraved grid patterns. This grid pattern, leaving a negative imprint on thin-layer embedded samples, allows identification of cells selected from the time-lapse imaging for serial-section electron microscopy. This method facilitates the ultrastructural analysis of specific stages of abscission.
Methods in cell biology 01/2010; 96:591-601. · 2.05 Impact Factor
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ABSTRACT: During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.
The Journal of Cell Biology 12/2003; 163(3):451-6. · 10.26 Impact Factor
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ABSTRACT: HeLa cells are widely used as a model system to study cell division. The last step of cell division, abscission, occurs at an about 1 μm wide intercellular bridge that connects the post-mitotic sister cells. Abscission often occurs long after ingression of the cleavage furrow, and no efficient methods to synchronize cells to this stage are available. Here, we have developed a correlative fluorescence time-lapse imaging and electron microscopic approach using Aclar sheets with engraved grid patterns. This grid pattern, leaving a negative imprint on thin-layer embedded samples, allows identification of cells selected from the time-lapse imaging for serial-section electron microscopy. This method facilitates the ultrastructural analysis of specific stages of abscission.
Methods in Cell Biology.
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ABSTRACT: The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.
Journal of Microscopy, v.230, 297-307 (2008).
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Sider Penkov,
Fanny Mende,
Vyacheslav Zagoriy,
Cihan Erkut,
René Martin,
Ulrike Pässler,
Kai Schuhmann,
Dominik Schwudke,
Margit Gruner, Jana Mäntler,
Thomas Müller-Reichert,
Andrej Shevchenko,
Hans-Joachim Knölker,
Teymuras V Kurzchalia
Angewandte Chemie (International ed. in English), v.49, 9430-9435 (2010).
