Flattening <i>Drosophila</i> cells for high‐resolution light microscopic studies of mitosis in vitro

Fleming, Shawna L.; Rieder, Conly L.

doi:10.1002/cm.10143

Cited by 24 publications

(18 citation statements)

References 30 publications

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“…For analysis of mitotic progression in live Drosophila neuroblasts, brains from third instar larva were dissected and processed as described previously (Fleming and Rieder, 2003;Savoian and Rieder, 2002) with the following modifications. The dissected brain was put on a coverslip with a small drop of neuroblast culture medium supplemented with 20% FBS.…”

Section: Time-lapse Dic Microscopy Of Neuroblastsmentioning

confidence: 99%

Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila

Williams

Leung

Maiato

et al. 2007

Journal of Cell Science

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We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components. Animals carrying mutations in mitch die as late third-instar larvae; mitotic neuroblasts in larval brains exhibit high levels of aneuploidy. Analysis of fixed D. melanogaster brains and mitch RNAi in cultured cells, as well as video recordings of cultured mitch mutant neuroblasts, reveal that chromosome alignment in mitch mutants is compromised during spindle formation, with many chromosomes displaying persistent mono-orientation. These misalignments lead to aneuploidy during anaphase. Mutations in mitch also disrupt chromosome behavior during both meiotic divisions in spermatocytes: the entire chromosome complement often moves to only one spindle pole. Mutant mitotic cells exhibit contradictory behavior with respect to the spindle assembly checkpoint (SAC). Anaphase onset is delayed in untreated cells, probably because incorrect kinetochore attachment maintains the SAC. However, mutant brain cells and mitch RNAi cells treated with MT poisons prematurely disjoin their chromatids, and exit mitosis. These data suggest that Mitch participates in SAC signaling that responds specifically to disruptions in spindle microtubule dynamics. The mitch gene corresponds to the transcriptional unit CG7242, and encodes a protein that is a possible ortholog of the Spc24 or Spc25 subunit of the Ndc80 kinetochore complex. Despite the crucial role of Mitch in cell division, the mitch gene has evolved very rapidly among species in the genus Drosophila.

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Section: Time-lapse Dic Microscopy Of Neuroblastsmentioning

confidence: 99%

Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila

Williams

Leung

Maiato

et al. 2007

Journal of Cell Science

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“…One major disadvantage though, has been related with the fact that most Drosophila cell lines are semi-adherent and thus cells are round, making highresolution microscopy difficult. To overcome this problem we have modified the agaroverlay technique recently described for Drosophila culture cells by Fleming and Rieder (2003) (17) so that we can control the degree of cell flattening. Unlike cells flattened by growing on a concanavalin A substrate (18), which is particularly useful for routine microscopy studies, the agar overlay approach allows one to select cells …”

Section: Introductionmentioning

confidence: 99%

Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells

Pereira

Matos

Lince-Faria

et al. 2009

Methods in Molecular Biology

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Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (like newt lung cells, PtK1 cells or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference

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“…Two major limitations of this method were that neuroblasts deteriorated relatively quickly in culture (within less than 2 h) and that wild-type neuroblasts contained secondary spindles besides the primary main spindle (in 17% of all observations) or failed to complete cytokinesis (Savoian and Rieder, 2002). Using a modified technique, Fleming and Rieder (2003) were able to prolong viability of neuroblasts in culture, but cytokinesis defects still occurred. We improved the original technique of Savoian and Rieder by making two essential changes: 1) minimizing physiological stress by substituting Voltalef oil with insect culture media, and 2) minimizing physical stress by imaging neuroblasts in whole larval brain explants instead of brain squashes (see Materials and Methods).…”

Section: Time-lapse Analysis Of the Cell Cycle In Larval Brain Neurobmentioning

confidence: 99%

Live Imaging ofDrosophilaBrain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control

Siller

Serr

Steward

et al. 2005

MBoC

120

132

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Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactindependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.

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Flattening Drosophila cells for high‐resolution light microscopic studies of mitosis in vitro

Cited by 24 publications

References 30 publications

Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila

Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila

Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells

Live Imaging ofDrosophilaBrain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control

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