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A single Drosophila embryo extract for the study of mitosis ex vivo

Nature Protocols volume 8, pages 310–324 (2013)Cite this article

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Abstract

Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60–90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.

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Figure 1: Pipette-based extraction system on an inverted light microscope.
Figure 2: Flowchart of working steps for embryo extract preparation.
Figure 3: Embryo preparation and extraction procedure.
Figure 4: Example of nuclear divisions in extracts.

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Acknowledgements

We thank the EMBL Mechanical Workshop for equipment construction, and the Bloomington Stock Center for fly stocks. We thank E.M. Tranfield (EMBL) for suggesting correlative light-electron microscopy as an application. I.A.T. was a recipient of an Advanced Researcher Fellowship from the Swiss National Science Foundation (SNF), and acknowledges additional support from the EMBL. I.G. was a recipient of an EMBO long-term fellowship and an EMBL EIPOD fellowship. A.E. acknowledges support of the EMBL. T.S. acknowledges support of the EMBL and Cancer Research UK. This work was supported by the EMBL.

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Author notes

  1. Anne Ephrussi and Thomas Surrey: These authors jointly directed this work.

Authors and Affiliations

  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Ivo A Telley & Thomas Surrey

  2. Developmental Biology Unit, EMBL, Heidelberg, Germany

    Ivo A Telley, Imre Gáspár & Anne Ephrussi

  3. Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London, UK

    Thomas Surrey

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  1. Ivo A Telley
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Contributions

I.A.T. and I.G. determined the correct timing of extraction. I.A.T. designed the instrument, drew up the protocol and performed the experiments. I.A.T. and T.S. conceived the cycle arrest experiment. I.A.T. prepared the figures. A.E. and T.S. supervised the work. All authors discussed the work and contributed to the writing of the manuscript.

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Correspondence to Ivo A Telley.

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Telley, I., Gáspár, I., Ephrussi, A. et al. A single Drosophila embryo extract for the study of mitosis ex vivo. Nat Protoc 8, 310–324 (2013). https://doi.org/10.1038/nprot.2013.003

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